Methods and compositions for rejuvenating cns glial populations by suppresion of transcription factors

ABSTRACT

The present disclosure is directed to methods of inducing rejuvenation in a population of adult glial progenitor cells, and methods of treating a subject having a myelin deficiency. The method of inducing rejuvenation in a population of adult glial progenitor cells may comprise administering, to the population of adult glial progenitor cells, an effective amount of an agent that suppresses one or more transcription factors selected from the group consisting of (i) zinc finger protein 274 (ZNF274), (ii) Myc-associated factor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos (IKZF3), and (v) signal transducer and activator of transcription 3 (STAT3).

This application claims priority from U.S. provisional Nos. 63/257,827,filed Oct. 20, 2021, and 63/350,039, 63/350,041, 63/350,042, filed Jun.8, 2022, which are incorporated herein by reference.

This invention was made with government support under NS110776 andAG072298 awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD

This application relates to methods and compositions for rejuvenatingCNS glial populations with an agent suppressing a transcription factor.

BACKGROUND

Glial progenitor cells (GPCs, also referred to as oligodendrocyteprogenitor cells and NG2 cells) colonize the human brain duringdevelopment, and persist in abundance throughout adulthood. Duringdevelopment, human GPCs (hGPCs) are highly proliferative bipotentialcells, producing new oligodendrocytes and astrocytes (French-Constantand Raff, “Proliferating Bipotential Glial Progenitor Cells in Adult RatOptic Nerve,” Nature 319:499-502 (1986) and Raff et al., “A GlialProgenitor Cell that Develops in Vitro into an Astrocyte or anOligodendrocyte Depending on Culture Medium,” Nature 303:390-396(1983)). In rodents, this capacity wanes during normal aging, withproliferation, migration, and differentiation competence all diminishingin aged GPCs (Chari et al., “Decline in Rate of Colonization ofOligodendrocyte Progenitor Cell (OPC)-depleted Tissue by Adult OPCs WithAge,” J. Neuropathol. Exp. Neurol. 62:908-916 (2003); Gao and Raff,“Cell Size Control and a Cell-intrinsic Maturation Program inProliferating Oligodendrocyte Precursor Cells,” J. Cell Biol.138:1367-1377 (1997); Moyon et al., “TET1-mediated DNAHydroxymethylation Regulates Adult Remyelination in Mice,” NatureCommunications 12:3359-3359 (2021); Segel et al., “Niche StiffnessUnderlies the Ageing of Central Nervous System Progenitor Cells,” Nature573:130-134 (2019); Tang et al., “Long-Term Culture of PurifiedPostnatal Oligodendrocyte Precursor Cells. Evidence for an IntrinsicMaturation Program that Plays Out Over Months,” J. Cell Biol.148:971-984 (2000); Temple & Raff, “Clonal Analysis of OligodendrocyteDevelopment in Culture: Evidence for a Developmental Clock that CountsCell Divisions,” Cell 44:773-779 (1986); Wolswijk & Noble,“Identification of an Adult-Specific Glial Progenitor Cell,” Development105:387-400 (1989); and Wren et al., “In Vitro Analysis of the Originand Maintenance of O-2Aadult Progenitor Cells,” J. Cell Biol.116,:167-176 (1992)). Similarly, it was previously found that adulthuman GPCs are less proliferative, less migratory, and more readilydifferentiated than their fetal counterparts when transplanted intocongenitally dysmyelinated murine hosts (Windrem et al., “Fetal andAdult Human Oligodendrocyte Progenitor Cell Isolates Myelinate theCongenitally Dysmyelinated Brain,” Nat. Med. 10:93-97 (2004)). Yetdespite the manifestly different competencies of fetal and adult hGPCs,and the abundant data on GPC transcription in rodent models of aging(Bouhrara et al., “Evidence of Demyelination in Mild CognitiveImpairment and Dementia Using a Direct and Specific Magnetic ResonanceImaging Measure of Myelin Content,” Alzheimers Dement. 14:998-1004(2018); de la Fuente et al., “Changes in the Oligodendrocyte ProgenitorCell Proteome with Ageing,” Mol. Cell Proteomics 19:1281- 1302 (2020);Neumann et al., “Metformin Restores CNS Remyelination Capacity byRejuvenating Aged Stem Cells,” Cell Stem Cell 25:473-485 e478 (2019);and Spitzer et al., “Oligodendrocyte Progenitor Cells Become RegionallyDiverse and Heterogeneous with Age,” Neuron 101:459-471 e455 (2019)),little data are available that address changes in GPC gene expressionduring human aging (Perlman et al., “Developmental Trajectory ofOligodendrocyte Progenitor Cells in the Human Brain Revealed by SingleCell RNA Sequencing,” Glia 68:1291-1303 (2020) and Sim et al.,“Complementary Patterns of Gene Expression by Human OligodendrocyteProgenitors and their Environment Predict Determinants of ProgenitorMaintenance and Differentiation,” Ann. Neurol. 59:763-779 (2006)), orthat provide clear head-to-head comparisons of transcription by fetaland adult human GPCs.

The present disclosure is directed to overcoming deficiencies in theart.

SUMMARY

One aspect of the present application relates to a method of inducingrejuvenation in a population of adult glial progenitor cells. The methodcomprises the step of administering, to the population of adult glialprogenitor cells, an effective amount of an agent that suppresses one ormore transcription factors selected from the group consisting of (i)zinc finger protein 274 (ZNF274), (ii) Myc-associated factor X (MAX),(iii) E2F transcription factor 6 (E2F6), (iv) zinc finger protein Aiolos(IKZF3), and (v) signal transducer and activator of transcription 3(STAT3).

Another aspect of the present application relates to a method oftreating a subject having a glial cell-related disorder. The methodcomprises the step of administering, to the subject, an effective amountof an agent that suppresses one or more transcription factors selectedfrom the group consisting of ZNF274, MAX, E2F6, IKZF3, and STAT3.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or application with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows Bulk RNA-Seq Characterization of human fetal GPCs. Panel A.Workflow of bulk and scRNA-Sequencing of CD140a+ , CD140a−, andA2B5+/PSA-NCAM−-selected 2nd trimester human fetal brain isolates. PanelB. Principal component analysis of all samples across two batches. PanelC. Venn diagram of CD140a+ vs CD140a− and CD140+ vs A2B5+/PSA-NCAM−differentially-expressed gene sets (p<0.01 and absolute log2-foldchange >1). Panel D. Significant Ingenuity Pathway Analysis terms forboth genesets. Size represents −log10 p-value and color representsactivation Z-Score (Blue, CD140a+; Red, A2B5+ or CD140a−). Panel E.Log2-fold changes of significant genes for both genesets. Missing barswere not significant. Panel F. Heatmap of transformed transcripts permillion (TPM) of selected genes in Panel E.

FIG. 2 shows single cell RNA-sequencing of CD140a and A2B5 selectedhuman fetal GPCs. Panel A. UMAP plot of the primary cell typesidentified during scRNA-Seq analysis of FACS isolated hGPCs derived from20 week gestational age human fetal VZ/SVZ. Panel B-Panel C. UMAP ofonly PSA-NCAM−/A2B5+ (B) or CD140a+ (C) human fetal cells. Panel D.Violin plots of cell type-selective marker genes. Panel E. Volcano plotof GPC vs pre-GPC populations. Panel F. Feature plots of selectdifferentially expressed genes between GPCs and pre-GPCs. Panel G.Select significantly-enriched GPC and pre-GPC IPA terms, indicatingtheir −log10 p-value and activation Z-Score. Panel H. Select featureplots of transcription factors predicted to be significantly activatedin fetal hGPCs. Relative transcription factor regulon activation isdisplayed as calculated using the SCENIC package.

FIG. 3 shows adult human GPCs are transcriptionally and functionallydistinct from fetal GPCs. Panel A. Workflow of bulk RNA-Seq analysis ofhuman adult and fetal GPCs. Panel B. Principal component analysis of allsamples across three batches. Panel C. Venn Diagram of both Adult vsFetal differential expression gene sets. Panel D. IPA network of curatedterms and genes. Node size is proportionate to node degree. Label colorcorresponds to enrichment in either adult (red) or fetal (blue)populations. Panel E. Bar plots of significant IPA terms by module.Z-Scores indicate predicted activation in fetal (blue) or adult (red)hGPCs. Panel F. Bar plot of log2-fold changes and heatmap of networkgenes' TPM.

FIG. 4 shows inference of transcription factor activity implicates a setof transcriptional repressors in the establishment of adult hGPCidentity. Panel A. Normalized enrichment score plots of significantlyenriched transcription factors predicted to be active in fetal and adultGPCs. Each dot is a motif whose size indicates how many genes in whichthat motif is predicted to be active, and the color represents thewindow around the promoter at which that motif was found enriched. PanelB. Heatmap of enriched TF TPMs, and C, log-fold changes vs adult GPCs,for both fetal hGPC isolates. Panel D-Panel G. Predicted directtranscription factor activity of curated genes split into D fetalactivators; Panel E, fetal repressors; Panel F, adult activators; andPanel G, adult repressors. Color indicates differential expression ineither adult (red) or fetal (blue) hGPCs; shape dictates type of node(octagon, repressor; rectangle, activator; oval, other target gene).Boxed and circled genes indicate functionally-related genes contributingto either glial progenitor/oligodendrocyte identity,senescence/proliferation targets, or upstream or downstream TFs thatwere also deemed activated.

FIG. 5 shows induction of an aged GPC transcriptome via adulthGPC-enriched repressors. Panel A. Schematic outlining the structure offour distinct doxycycline (Dox)-inducible EGFP lentiviral expressionvectors, each encoding one of the transcriptional repressors: E2F6,IKZF3, MAX, or ZNF274. Panel B. Induced pluripotent stem cell(iPSC)-derived hGPC cultures (line C27 (Chambers et al., Naturebiotechnology, 27, 275-280, 2009; Wang et al., Cell Stem Cell 12,252-264, 2013)) were transduced with a single lentivirus or vehicle forone day, and then treated with Dox for the remainder of the experiment.At 3, 7, and 10 days following initiation of Dox-induced transgeneexpression, hGPCs were isolated via FACS for qPCR. Panel C. qPCRs ofDox-treated cells showing expression of each transcription factor, vsmatched timepoint controls. Panel D. qPCR fold-change heatmap of selectaging related genes. Within timepoint comparisons to controls werecalculated via post hoc least-squares means tests of linear modelsfollowing regression of a cell batch effect. FDR adjusted p-values:*<0.05, **<0.01, ***<0.001.

FIG. 6 shows miRNAs drive adult GPC transcriptional divergence inparallel to transcription factor activity. Panel A. Principal componentanalysis of miRNA microarray samples from human A2B5+ adult and CD140a+fetal GPCs. Panel B. Log2 fold change bar plots and heatmap ofdifferentially expressed miRNAs. Panel C. Characterization bubble plotof enrichment of miRNAs, versus the average log2 FC of its predictedgene targets. Panel D-Panel E. Curated signaling networks of Panel D,fetal (top) and Panel E, adult (bottom) enriched miRNAs and theirpredicted targets.

FIG. 7 , which is related to FIG. 1 , shows enrichment of human fetalGPCs via CD140a+ or A2B5+/PSA-NCAM− selection: Panel A. Principalcomponent analysis of CD140a+ and A2B5+fetal GPCs. Panel B. Volcanoplots indicating significant A2B5 (Green) and CD140a (Blue) enrichedgenes. Panel C. Principal component analysis of CD140a+ and CD140a−fetal cells. Panel D. Volcano plots indicating significant CD140a−(Magenta) and CD140a (Blue) enriched genes. Panel E. Upset plot ofsignificant up and downregulated genes in both genesets.

FIG. 8 , which is related to FIG. 2 , shows single cell RNA-Seq qualityfiltering: Violin plots of unfiltered Panel A. A2B5+/PSA-NCAM− and PanelB. CD140a scRNA-seq captures. Panel C-D, Violin plots following qualityfiltration (Percent mitochondrial gene expression<15% and >500 uniquegenes) of Panel C, A2B5+/PSA-NCAM− and Panel D, CD140a+ captures.

FIG. 9 , which is related to FIG. 2 , shows single cell RNA-sequencingof PSA-NCAM−/A2B5+ vs CD140a+ fetal hGPCs: Panel A. UMAP plot of A2B5+and CD140a+ fetal hGPCs. Panel B. Frequency of cell types in eachsorting paradigm isolate. Panel C. Scatter plot of differentiallyexpressed bulk RNA-Seq log2 fold changes vs pseudobulk log2 fold changesbetween CD140a+ and A2B5+ fetal hGPC isolates.

FIG. 10 , which is related to FIG. 4 , shows shared motifs of activetranscription factors in fetal or adult hGPCs: matrix of all predictedactive transcription factors in fetal and adult GPCs. Size and colorindicate degree of motifs that are shared between transcription factors.

FIG. 11 , which is related to FIG. 5 , shows adult repressor isoformexpression. Bar plots of transcripts per million (TPMs) of all proteincoding adult repressor isoforms in each GPC group.

FIG. 12 , which is related to FIG. 5 , shows bulk RNA-Seq ofiPSC-derived hGPCs reveals concordant abundance of age-associated genes.iPSC-derived hGPCs (C27) were isolated via CD140a+ FACS and assayed viabulk RNA-sequencing. Abundance of relevant glial age-associated genes,including those in our active transcription factor cohort, are displayedalongside fetal and adult hGPC data.

FIG. 13 , which is related to FIG. 6 , shows transcription factorregulation of miRNAs provides post-transcriptional modulation of glialaging gene expression: Panel (A) Log2 FC violin plots of significantadult vs fetal GPC transcription factors predicted to be upstream ofdifferentially expressed adult vs fetal GPC miRNAs. Panel (B) Network ofidentified transcription factors from FIG. 2 and their predictedregulation of differentially expressed adult vs fetal hGPC miRNAs.

FIG. 14 demonstrates: Panel A. Design of the CBh-BCL11 A-GFP and controlCBh-GFP lentivirus. Panel B: Generation of CRISPR-Cas9-modified C27 iPSCline expressing tRFP-mScarlet from the AAVS1 locus. Panel C. Schematicdepicting the experimental paradigm for long-term chimerization and BCL11 A overexpression in Rag1 mice. Panel D. BCL11A overexpression viaCBh-BCL11A-GFP confirmed in vitro by ddPCR and (Panel E) in vivo byimmunohistochemistry.

FIG. 15 demonstrates: Panel A. A coronal section of a Rag1-C271RFP mousecorpus callosum (CC) three weeks after injection with CBh-BCL11A-GFP (L)or CBH-GFP (R). Panel B. Zoomed-in coronal sections of the CC, showingthe difference in RFP-tagged human cells and OLIG2 between BCL11A-GFPand GFP-only hemispheres, quantified in Panel C). Panel D. Magnifiedcoronal sections of the CC, showing the difference in RFP-lagged humancells and mouse NG2 between BCL11A-GFP and GFP-only hemispheres,quantified in Panel E).

FIG. 16 demonstrates: Panel A. Coronal images of chimeric mouse corpuscallosum (CC) three weeks after injection with CBh-BCL11A-GFP)L_ orCBh-GFP (R). Human cells are marked with tRFP/mScarlet and human nuclearantigen (hNA) in red, and OLIG2 is stained in green. Panel B. Coronalimages of chimeric mouse CC six weeks after injection, human cellsmarked with tRFP/mScarlet and hNA in red, and OLIG2 in green. Panel C)Coronal images of chimeric mouse CC three weeks after injection, humancells marked with tRFP/mScarlet and hNA in red, and PDGFRa in green.Panel D. Coronal images of chimeric mouse CC three weeks afterinjection, human cells marked with tRFP/mScarlet and hNA in red, andmouse NG2 in green.

FIG. 17 shows an exemplary design of a BCL11A expression vector.

DETAILED DESCRIPTION

Reference will be made in detail to certain aspects and exemplaryembodiments of the application, illustrating examples in theaccompanying structures and figures. The aspects of the application willbe described in conjunction with the exemplary embodiments, includingmethods, materials and examples, such description is non-limiting, andthe scope of the application is intended to encompass all equivalents,alternatives, and modifications, either generally known, or incorporatedhere. The described aspects, features, advantages, and characteristicsof the invention may be combined in any suitable manner in one or morefurther embodiments. One skilled in the relevant art will recognize thatthe invention may be practiced without one or more of the specificaspects or advantages of a particular embodiment. In other instances,additional aspects, features, and advantages may be recognized andclaimed in certain embodiments that may not be present in allembodiments of the invention. Further, one skilled in the art willrecognize many techniques and materials similar or equivalent to thosedescribed here, which could be used in the practice of the aspects andembodiments of the present application. The described aspects andembodiments of the application are not limited to the methods andmaterials described.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a peptide”includes “one or more” peptides or a “plurality” of such peptides.

I. Definitions

As used herein, the following terms or phrases (in parentheses) shallhave the following meanings:

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a peptide”includes “one or more” peptides or a “plurality” of such peptides.

The term “about” or “approximately” includes being within astatistically meaningful range of a value. Such a range can be within anorder of magnitude, preferably within 50%, more preferably within 20%,still more preferably within 10%, and even more preferably within 5% ofa given value or range. The allowable variation encompassed by the term“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, and so on. As a non-limiting example, each range discussedherein can be readily broken down into a lower third, middle third andupper third, and so on. As will also be understood by one skilled in theart all language such as “up to,” “at least,” and the like include thenumber recited and refer to ranges which can be subsequently broken downinto subranges as discussed above. Finally, as will be understood by oneskilled in the art, a range includes each individual member.

In understanding the scope of the present application, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “involving”, “having”,and their derivatives. The term “consisting” and its derivatives, asused herein, are intended to be closed terms that specify the presenceof the stated features, elements, components, groups, integers, and/orsteps, but exclude the presence of other unstated features, elements,components, groups, integers and/or steps. The term “consistingessentially of”, as used herein, is intended to specify the presence ofthe stated features, elements, components, groups, integers, and/orsteps as well as those that do not materially affect the basic and novelcharacteristic(s) of features, elements, components, groups, integers,and/or steps. In embodiments or claims where the term comprising (or thelike) is used as the transition phrase, such embodiments can also beenvisioned with replacement of the term “comprising” with the terms“consisting of” or “consisting essentially of” The methods, kits,systems, and/or compositions of the present disclosure can comprise,consist essentially of, or consist of, the components disclosed.

In embodiments comprising an “additional” or “second” component, thesecond component as used herein is different from the other componentsor first component. A “third” component is different from the other,first, and second components, and further enumerated or “additional”components are similarly different.

The term “complementary” when used in connection with nucleic acid,refers to the pairing of bases, A with T or U, and G with C. The term“complementary” refers to nucleic acid molecules that are completelycomplementary, that is, form A to T or U pairs and G to C pairs acrossthe entire reference sequence, as well as molecules that are partially(e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) complementary.

The terms “nucleic acid”, “nucleotide”, and “polynucleotide” encompassboth DNA and RNA unless specified otherwise.

The term “polypeptide,” “peptide” or “protein” are used interchangeablyand to refer to a polymer of amino acid residues. The terms encompassall kinds of naturally occurring and synthetic proteins, includingprotein fragments of all lengths, fusion proteins and modified proteins,including without limitation, glycoproteins, as well as all other typesof modified proteins (e.g., proteins resulting from phosphorylation,acetylation, myristoylation, palmitoylation, glycosylation, oxidation,formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation,biotinylation, etc.).

The terms “abrogate”, “abrogation” “eliminate”, or “elimination” ofexpression of a gene or gene product (e.g., RNA or protein) refers to acomplete loss of the transcription and/or translation of a gene or acomplete loss of the gene product (e.g., RNA or protein). Expression ofa gene or gene product (e.g., RNA or protein) can be detected bystandard art known methods such as those described herein, as comparedto a control, e.g., an unmodified cell.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become produced, for exampleproducing an RNA or a protein by activating the cellular functionsinvolved in transcription and/or translation of a corresponding gene orDNA sequence. A DNA sequence is expressed in or by a cell to form an“expression product” such as an RNA or a protein. The expression productitself, e.g., the resulting protein, may also be said to be “expressed”by the cell. An expression product can be characterized asintracellular, extracellular or transmembrane

As used herein, the term “glial cells” refers to a population ofnon-neuronal cells that provide support and nutrition, maintainhomeostasis, either form myelin or promote myelination, and participatein signal transmission in the nervous system. “Glial cells” as usedherein encompasses fully differentiated cells of the glial lineage, suchas oligodendrocytes or astrocytes, as well as glial progenitor cells,each of which can be referred to as macroglial cells.

As used herein, the term “adult glial progenitor cells” refers to glialprogenitor cells that are present in a mammal at any developmental stageafter birth. In some embodiments, the term “adult glial progenitorcells” refers to glial progenitor cells present in a human subject whois 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18years of age or older. In some embodiments, the term “adult glialprogenitor cells” refers to glial progenitor cells present in a humansubject who is 20 years of age or older, 25 years of age or older, 30years of age or older, 35 years of age or older, 40 years of age orolder, 45 years of age or older, or 50 years of age or older. In someembodiments, the term “adult glial progenitor cells” refers to glialprogenitor cells present in a human subject of advanced age, such as anadult of 55 years of age or older, 60 years of age or older, 65 years ofage or older, 70 years of age or older, 75 years of age or older, or 80years of age or older.

The term “a functional variant” of a gene product (e.g., a transcriptionfactor), refers to a modified transcription factor (e.g., by deletion,substitution, insertion, glycosylation, etc.) that retains at least 50%of the biological activity of the unmodified (wild-type) transcriptionfactor in a competition assay.

The term “effective amount” refers to the amount of active compound orpharmaceutical agent that elicits the biological or medicinal responsein a tissue, system, animal, individual or human that is being sought bya researcher, veterinarian, medical doctor or other clinician.

The term “regulatory sequence” or “regulatory element” refers to thenucleic acid sequences or elements that control, regulate, cause orpermit expression of a gene to be regulated by such regulatory sequenceor element. Regulatory elements/sequences may be found at the 5′ or 3′side of the coding region, or within the coding region, or withinintrons, of the gene to be regulated. Examples of regulatorysequences/elements include, but are not limited to, promoters,enhancers, RNA polymerase initiation sites, ribosome binding sites, andother sequences that facilitate the expression of encoded polypeptidesin a given expression system

The term “promoter”, as used herein, refers to a nucleotide sequencecapable of controlling the expression of a coding sequence or functionalRNA. In general, the polynucleotide of interest is located 3′ of apromoter sequence. In some embodiments, the promoter is derived in itsentirety from a native gene. In some embodiments, the promoter iscomposed of different elements derived from different naturallyoccurring promoters. In some embodiments, the promoter comprises asynthetic nucleotide sequence. It will be understood by those skilled inthe art that different promoters will direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions or to the presenceor the absence of a drug or transcriptional co-factor. Ubiquitous,cell-type-specific, tissue-specific, developmental stage-specific, andconditional promoters, for example, drug-responsive promoters (e.g.tetracycline-responsive promoters) are well known to those of skill inthe art. Examples of promoter include, but are not limited to, thephosphoglycerate kinase (PKG) promoter, CAG, NSE (neuronal specificenolase), synapsin or NeuN promoters, the SV40 early promoter, mousemammary tumor virus LTR promoter; adenovirus major late promoter (AdMLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV)promoter such as the CMV immediate early promoter region (CMVIE), SFFVpromoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybridpromoters, and the like. The promoters can be of human origin or fromother species, including from mice. In addition, sequences derived fromnonviral genes, such as the murine metallothionein gene promoter, willalso find use herein. In some embodiments, the promoter is aheterologous promoter. In some embodiments, a promoter sequence consistsof proximal and more distal upstream elements and can comprise anenhancer element.

The term “heterologous promoter”, as used herein, refers to a promoterthat does is not found to be operatively linked to a given encodingsequence in nature.

The term “enhancer” refers to a nucleotide sequence that can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter.

The term “operatively linked” or “operably linked” refers to theassociation of two or more nucleic acid fragments on a single nucleicacid fragment so that the function of one is affected by the other. Forexample, a promoter is operatively linked with a coding sequence when itis capable of affecting the expression of that coding sequence (e.g.,the coding sequence is under the transcriptional control of thepromoter). Encoding sequences can be operatively linked to regulatorysequences in sense or antisense orientation.

The term “transcription factor” refers to a DNA-binding protein thatregulate the expression of specific genes.

A transcription factor can have a positive effect on gene transcriptionand, thus, may be referred to as a “transcription activator,”“activator” or a “transcriptional activation factor.” An exemplaryactivator (or transcriptional activation factor) is signal transducerand activator of transcription 3 (STAT3). As illustrated in FIG. 4F ofthe present disclosure, in the context of adult glial progenitor cells,STAT3 is predicted to activate a set of senescence-associated genes(e.g., BIN1, DMTF1, CD47, CTNNA1, RUNX2, RUNX1, MAP3K7, and OGT), glialcell-associated genes (e.g., PLP1, CNP, PMP22, SEMA4D, CLDN11, GPR37,MYRF, MAG, BCAS1, ST18, ERBB4, CERS2, LPAR1, and GJB1), and downstreamtranscription factors (e.g., MAX, E2F6, and IKZF3).

A transcription factor can also negatively affect gene expression and,thus, may be referred to as “transcription repressor,” “repressor” or a“transcription repression factor.” Exemplary repressors (ortranscription repression factors) involved in glial progenitor cellssenescence include, without limitation, ZNF274, MAX, E2F6, and IKZF3. Asillustrated in FIG. 4G of the present disclosure, in the context ofadult glial progenitor cells, ZNF274, MAX, E2F6, and IKZF3 are predictedto repress sets of proliferation-associated gene targets (e.g., YAP1,LMNB1, PATZ1, TEAD1, FN1, TP53, CDK1, CCND2, CDKN2D, CENPH, MKI67, CDK4,CENPF, CDK5, CDKN3, and CHEK1), glial cell- associated genes (e.g.,CHRDL1, ST8SIA1, PTPRZ1, CA10, PDGFRA, BCAN, NXPH1, CSPG4, and, PCDH15),and downstream transcription factors (e.g., BLC11A, EZH2, HDAC2, NF1B,MYC, HMGA2, and TEAD).

The term “inhibitor of transcription factor” or “transcription factorrepressor” refers to an agent that inhibits activity or expression of atranscription factor. An “inhibitor of transcription factor ” or“transcription factor repressor” may be a small molecule, a polypeptide,a polynucleotide, such as an antisense oligonucleotide (ASO), a shRNA,or a miRNA.

Certain terms employed in the specification, examples, and claims arecollected herein. Unless defined otherwise, all technical and scientificterms used in this disclosure have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs.

Preferences and options for a given aspect, feature, embodiment, orparameter of the disclosure should, unless the context indicatesotherwise, be regarded as having been disclosed in combination with anyand all preferences and options for all other aspects, features,embodiments, and parameters of the disclosure.

II. Methods Involving the Activation of Transction Factors

One aspect of the present application relates to a method of inducingrejuvenation in adult glial progenitor cells by activating certaintranscription factors. In some embodiments, the method involvesexpressing, in the adult glial progenitor cells, an effective amount ofone or more transcription factors selected from the group consisting ofB-cell lymphoma/leukemia 11A (BCL11A), histone deacetylase 2 (HDAC2),histone-lysine N-methyltransferase EZH2 (EZH2), myc proto-oncogeneprotein (MYC), high 15 mobility group protein HMGI-C (HMGA2), nuclearfactor 1 B-type (NFIB) and transcriptional enhancer factor TEF-4(TEAD2).

As described herein, the term “rejuvenation” or “rejuvenating” refers toa reversion of the aging process in a cell and a return to youthful cellstate, in particular with regard to proliferative and/or differentiationcapacity, without loss of cell identity

Adult glial progenitor cells suitable for use in the methods disclosedherein include mammalian glial progenitor cells, e.g., human glialprogenitor cells, rodent glial progenitor cells, non-human primate glialprogenitor cells, ovine glial progenitor cells, bovine glial progenitorcells, porcine glial progenitor cells, canine glial progenitor cells,and feline glial progenitor cells. In some embodiments, the adult glialprogenitor cells are adult human glial progenitor cells.

In some embodiments, the glial progenitor cells are adult human glialprogenitor cells. In some embodiments, the step of expressing isperformed ex vivo. In some embodiments, the step of expressing isperformed in vivo.

Another aspect of the application relates to a method of treating myelindeficiency in a subject by activating certain transcription factors inglial progenitor cells of the subject. In some embodiments, the methodinvolves expressing, in glial progenitor cells of the subject, aneffective amount of one or more transcription factors selected from thegroup consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2.

In some embodiments, the step of expressing is performed ex vivo. Insome embodiments, the step of expressing is performed in vivo.

In accordance with this aspect of the present application, the myelindeficiency may be associated with a condition selected from the groupconsisting of multiple sclerosis, neuromyelitis optica, transversemyelitis, optic neuritis, subcortical stroke, diabeticleukoencephalopathy, hypertensive leukoencephalopathy, age-related whitematter disease, spinal cord injury, radiation- or chemotherapy induceddemyelination, post-infectious and post-vaccinial leukoencephalitis,periventricular leukomalacia, pediatric leukodystrophy (e.g.,Pelizaeus-Merzbacher Disease, Tay-Sach Disease, Sandhoff'sgangliosidoses, Krabbe's disease, metachromatic leukodystrophy,mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy,Canavan's disease, Vanishing White Matter Disease, and AlexanderDisease), lysosomal storage diseases, congenital dysmyelination,inflammatory demyelination, vascular demyelination, and cerebral palsy.

In some embodiments, the myelin deficiency is associated with aneurodegenerative disease, e.g., Huntington's disease. As used herein,“Huntington's disease” refers to an autosomal dominant inherited braindisorder that typically becomes manifest in adulthood. Huntington'sdisease pathology is characterized by hypomyelination, as well asneuronal and white matter loss (see, e.g., Osipovitch et al., “HumanESC-Derived Chimeric Mouse Models of Huntington's Disease RevealCell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” CellStem Cell 24(1):107-122 (2019), which is hereby incorporated byreference in its entirety).

In other embodiments, the myelin deficiency is associated with aneuropsychiatric disease, e.g., schizophrenia. As used herein,“schizophrenia” refers to a condition typically characterized by arelative paucity of white matter and often frank hypomyelination (see,e.g., Windrem et al., “Human iPSC Glial Mouse Chimeras Reveal GlialContributions to Schizophrenia,” Cell Stem Cell 21(2):195-208 (2017),which is hereby incorporated by reference in its entirety).

As used hereinafter, the term, “treating” a subject having a myelindeficiency encompasses: (1) preventing, delaying, or reducing theincidence and/or likelihood of the appearance of at least one clinicalor sub-clinical symptom of the myelin deficiency developing in a subjectthat may be afflicted with or predisposed to the myelin deficiency, butdoes not yet experience or display clinical or subclinical symptoms ofthe myelin deficiency; or (2) inhibiting the myelin deficiency, i.e.,arresting, reducing or delaying the development of the myelin deficiencyor a relapse thereof or at least one clinical or sub-clinical symptomthereof; or (3) relieving the myelin deficiency, i.e., causingregression of the myelin deficiency or at least one of its clinical orsub-clinical symptoms. The benefit to a subject to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician.

As used hereinafter, the term “subject” refers to an individualorganism, for example, an individual mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal. In some embodiments, the subject is a non-human primate. In someembodiments, the subject is a rodent. In some embodiments, the subjectis a sheep, a goat, a cat, or a dog. In some embodiments, the subject isa research animal. In some embodiments, the subject is geneticallyengineered, e.g., a genetically engineered non-human subject. Thesubject may be an adult subject. In some embodiments, the subject is atleast 1 year old, least 2 year old, least 4 year old, least 6 year old,least 8 year old, least 10 year old, least 12 year old, least 15 yearold, at least 18 years old, at least 20 years old, at least 25 yearsold, at least 30 years old, at least 35 years old, at least 40 yearsold, at least 45 years old, at least 50 years old, at least 55 yearsold, at least 60 years old, at least 65 years old, at least 70 yearsold, at least 75 years old, at least 80 years old, at least 85 yearsold, at least 90 years old, at least 95 years old, at least 100 yearsold, or more. In some embodiments, the subject is an adult subjectbetween 18 to 100 years old, 20 to 100 years old, 30 to 100 years old,40 to 100 years old, 50 to 100 years old, 50 to 100 years old, 60 to 100years old, 70 to 100 years old, 80 to 100 years old, or 90 to 100 yearsold.

In some embodiments, the expressing step in the method of inducingrejuvenation in adult GPCs , or in the method of treating myelindeficiency in a subject, comprises administering to the adult GPCs orGPCs in the subject, respectively, one or more nucleic acid moleculesencoding one or more transcription factors selected from the groupconsisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2.

In some embodiments, the expressing step in the method of inducingrejuvenation in adult GPCs , or in the method of treating myelindeficiency in a subject, comprises administering to the adult GPCs orGPCs in the subject, respectively, one or more expression vectors thatexpress one or more transcription factors selected from the groupconsisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, whereineach of the expression vector comprises (1) a nucleotide sequenceencoding one or more transcription factors selected from the groupconsisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, and (2) aregulatory sequence operably linked to the nucleotide sequence.

As used herein, transcription factors mentioned in this application,such as BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, include alltranscript variants and functional variants thereof. Nucleotidesequences encoding the one or more transcription factors identifiedherein are well known and accessible in the art. Table 1 belowidentifies the transcription factors, and their transcript variants bytheir gene name, Gene ID No., and NCBI Reference transcript accessionnumber.

TABLE 1 Exemplary Human Genes and Transcript Variants Gene Reference IDTranscript Gene No.* Transcript Variant Accession Nos.* BAF chromatin53335 transcript variant 3 NM_138559.2 remodeling transcript variant 2NM_018014.4 complex subunit transcript variant 4 NM_001363864.1 (BCL11A)transcript variant X3 XM_011532910.1 transcript variant X1XM_011532909.1 transcript variant 1 NM_022893.4 transcript variant 5NM_001365609.1 transcript variant X7 XM_024452962.1 transcript variantX5 XM_017004335.1 transcript variant X2 XM_017004333.1 transcriptvariant X7 XM_024452963.1 transcript variant X8 XM_017004336.1 Histone3066 transcript variant 1 NM_001527.4 deacetylase 2 transcript variant 2NR_033441.2 (HDAC2) transcript variant 3 NR_073443.2 transcript variantX1 XM_017010799.1 Enhancer of 2146 transcript variant 1 NM_004456.5Zeste 2 polycomb transcript variant 2 NM_152998.3 repressive transcriptvariant 3 NM_001203247.2 complex 2 transcript variant 4 NM_001203248.2(EZH2) transcript variant 5 NM_001203249.2 transcript variant X3XM_005249962.4 transcript variant X6 XM_005249963.4 transcript variantX21 XM_005249964.4 transcript variant X2 XM_011515883.2 transcriptvariant X4 XM_011515884.2 transcript variant X5 XM_011515885.2transcript variant X7 XM_011515886.2 transcript variant X8XM_011515887.3 transcript variant X9 XM_011515888.2 transcript variantX11 XM_011515889.2 transcript variant X12 XM_011515890.2 transcriptvariant X13 XM_011515891.3 transcript variant X14 XM_011515892.2transcript variant X15 XM_011515893.2 transcript variant X17XM_011515894.2 transcript variant X19 XM_011515895.2 transcript variantX22 XM_011515896.2 transcript variant X23 XM_011515897.2 transcriptvariant X24 XM_011515898.2 transcript variant X26 XM_011515899.3transcript variant X30 XM_011515901.3 transcript variant X1XM_017011817.2 transcript variant X10 XM_017011818.1 transcript variantX18 XM_017011819.1 transcript variant X20 XM_017011820.2 transcriptvariant X25 XM_017011821.1 transcript variant X16 XM_024446680.1transcript variant X29 XR_001744581.1 transcript variant X27XR_002956413.1 transcript variant X28 XR_002956414.1 MYC proto- 4609transcript variant 1 NM_002467.6 oncogene, bHLH transcript variant 2NM_001354870.1 transcription factor (MYC) High mobility 8091 transcriptvariant 1 NM_003483.6 group AT hook 2 transcript variant 2 NM_003484.1(HMAG2) transcript variant 3 NM_001300918.1 transcript variant 4NM_001300919.1 transcript variant 5 NM_001330190.1 Nuclear 4781transcript variant 1 NM_001190737.2 Factor IB transcript variant 3NM_005596.3 (NFIB) transcript variant 2 NM_001190738.2 transcriptvariant 4 NM_001282787.2 transcript variant 5 NM_001369458.1 transcriptvariant 6 NM_001369459.1 transcript variant 7 NM_001369460.1 transcriptvariant 8 NM_001369461.1 transcript variant 9 NM_001369462.1 transcriptvariant 10 NM_001369463.1 transcript variant 11 NM_001369464.1transcript variant 12 NM_001369465.1 transcript variant 13NM_001369466.1 transcript variant 14 NM_001369467.1 transcript variant15 NM_001369468.1 transcript variant 16 NM_001369469.1 transcriptvariant 17 NM_001369470.1 transcript variant 18 NM_001369471.1transcript variant 19 NM_001369472.1 transcript variant 20NM_001369473.1 transcript variant 21 NM_001369474.1 transcript variant22 NM_001369475.1 transcript variant 23 NM_001369476.1 transcriptvariant 24 NM_001369477.1 transcript variant 25 NM_001369478.1transcript variant 26 NM_001369479.1 transcript variant 27NM_001369480.1 transcript variant 28 NM_001369481.1 transcript variant29 NR_161382.1 transcript variant 30 NR_161383.1 transcript variant 31NR_161384.1 transcript variant 32 NR_161385.1 transcript variant X1XM_005251467.3 transcript variant X14 XM_005251471.3 transcript variantX2 XM_006716773.3 transcript variant X5 XM_006716774.3 transcriptvariant X6 XM_006716775.3 transcript variant X9 XR_001746308.2transcript variant X10 XR_001746309.2 TEA Domain 8463 transcript variant3 NM_001256660.2 Transcription transcript variant 5 NM_003598.2 Factor 2transcript variant 1 NM_001256658.2 (TEAD2) transcript variant 2NM_001256659.2 transcript variant 4 NM_001256661.2 transcript variant 6NM_001256662.2 transcript variant X8 XM_005259334.4 transcript variantX7 XM_006723428.3 transcript variant X9 XM_006723429.2 transcriptvariant X1 XM_011527399.2 transcript variant X2 XM_011527400.2transcript variant X3 XM_011527401.1 transcript variant X4XM_011527402.2 transcript variant X5 XM_011527403.1 transcript variantX6 XM_011527404.2 transcript variant X10 XM_011527405.3 transcriptvariant XI1 XM_011527406.3 *Each of which is hereby incorporated byreference in its entirety.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding BCL11A, atranscript variant or functional variant thereof. In some embodiments,the one or more expression vectors comprises an expression vectorcomprising a nucleotide sequence encoding the BCL11A variant of SEQ IDNO:21. In some embodiments, the one or more expression vectors comprisesan expression vector comprising the nucleotide sequence of SEQ ID NO:20.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding HDAC2, atranscript variant or functional variant thereof.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding EZH2, atranscript variant or functional variant thereof.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding MYC, atranscript variant or functional variant thereof.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding HMGA2, atranscript variant or functional variant thereof.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding HFIB, atranscript variant or functional variant thereof.

In some embodiments, the one or more expression vectors comprises anexpression vector comprising a nucleotide sequence encoding TEAD2, atranscript variant or functional variant thereof.

In some embodiments, the expressing step in the method of inducingrejuvenation in adult GPCs, or in the method of treating myelindeficiency in a subject, comprises administering to the adult GPCs orGPCs in the subject, respectively, an effective amount of an inhibitorof a transcription factor repressor.

As used herein, the term “transcription factor repressor” refers to anagent that inhibits activity or expression of a transcription factor. A“transcription factor repressor” may be a polypeptide or apolynucleotide.

In some embodiments, the inhibitor of a transcription factor repressoris a small molecule.

In some embodiments, the inhibitor of a transcription factor repressoris a polypeptide or a polynucleotide. In some embodiments, theexpressing step in the method of inducing rejuvenation in adult GPCs, orin the method of treating myelin deficiency in a subject, comprisesadministering to the adult GPCs or GPCs in the subject, respectively, aneffective amount of an expression vector that comprises (1) a nucleotidesequence encoding an inhibitor of a transcription factor repressor, and(2) a regulatory sequence operably linked to the nucleotide sequence.

In some embodiments, the transcription factor repressor is aryl 20hydrocarbon receptor (AHR). In accordance with such embodiments, theinhibitor of transcription factor repressor is an AHR inhibitor. In someembodiments, the AHR inhibitor is a small molecule inhibitor.

Suitable AHR inhibitors are known in the art and include, withoutlimitation, a small molecule such as BAY-218 (see, e.g., Abstract 1288:Blocking Tumor Associated Immune Suppression with BAY-218, a Novel,Selective Aryl Hydrocarbon Receptor (AhR) Inhibitor,” Proceedings of theAmerican Association for Cancer 25 Research Annual Meeting 2019; 2019Mar. 29-Apr. 3; Atlanta, Ga. Philadelphia (Pa.): AACR; Cancer Res. 79(13Suppl):Abstract nr 1288 (2019), which is hereby incorporated byreference in its entirety); perillaldehyde (see, e.g., Fuyuno et al.,“Perillaldehyde Inhibits AHR Signaling and Activates NRF2 AntioxidantPathway in Human Keratinocytes,” Oxid. Med. Cell Longev. 2018:9524657(2018), which is hereby incorporated by reference in its entirety);StemRegenin 1 (SR1) (see, e.g., Boitano et al., “Aryl HydrocarbonReceptor Antagonists Promote the Expansion of Human Hematopoietic StemCells,” Science 329(5997):1345-1348 (2010), which is hereby incorporatedby reference in its entirety); KYN-101 (see, e.g., Campesato et al.,“Blockade of the AHR Restricts a Treg-Macrophage Suppressive AxisInduced by L-5 Kynurenine,” Nature Comm. 11:4011 (2020), which is herebyincorporated by reference in its entirety); CH-223191 (see, e.g., Kim etal., “Novel Compound 2-Methyl-2H-Pyrazole-3-Carboxylic acid(2-Methyl-4-o-Tolylazo-Phenyl)-Amide (CH-223191) Prevents2,3,7,8-TCDD-Induced Toxicity by Antagonizing the Aryl HydrocarbonReceptor,” Mol. Pharmaocl. 69(6):1871-1878 (2006), which is herebyincorporated by 10 reference in its entirety); BAY 2416964 (see, e.g.,International Patent Publication No. WO/2018/146010 to BayerAktiengesellschaft et al., which is hereby incorporated by reference inits entirety); PDM2 (see, e.g., de Medina et al., “Synthesis andBiological Properties of New Stilbene Derivatives of Resveratrol as NewSelective Aryl Hydrocarbon Modulators,” J. Med. Chem. 48:287-291 (2005),which is hereby 15 incorporated by reference in its entirety), andGNF351 (see, e.g., Smith et al., “Identification of a High-AffinityLigand that Exhibits Complete Aryl Hydrocarbon Receptor Antagonism,” J.Pharmacol. Exp. Ther. 338(1):318-327 (2011), which is herebyincorporated by reference in its entirety). Exemplary AHR small moleculeinhibitors are shown in Table 2 below.

TABLE 2 Exemplary AHR Small Molecule Inhibitors aryl hydro- carbon re-ceptor (AHR)

  BAY-218

  Perillaldehyde

  StemRegenim 1 (SR1)

  KYN-101

  CH-223191

  BAY 2416964

  PDM2

  GNF351

In some embodiments, the transcription factor repressor is Mycbox-dependent-interacting protein (BIN1). In accordance with suchembodiments, the inhibitor of a transcription factor repressor is a BIN1inhibitor.

In some embodiments, the transcription factor repressor is a miRNAmolecule. In accordance with such embodiments, the inhibitor of atranscription factor repressor is an inhibitor of miRNA. As describedherein, miRNAs that function as transcription factor repressors include,without limitation, those identified in Table 3 below.

TABLE 3 Exemplary Human miRNA Transcription factor  Repressors SEQ  ID miRNA Sequence NO: miR-193a-5p UGGGUCUUUGCGGGCGAGAUGA 1 miR-23b-3pAUCACAUUGCCAGGGAUUACCAC 2 miR-4687-3p UGGCUGUUGGAGGGGGCAGGC 3 miR-4651CGGGGUGGGUGAGGUCGGGC 4 miR-4270 UCAGGGAGUCAGGGGAGGGC 5 miR-24-3pUGGCUCAGUUCAGCAGGAACAG 6

In some embodiments, the inhibitor of a transcription factor repressoris an inhibitor of any one or more of the above identified miRNAs(target miRNAs). Inhibitors of miRNA are known in the art as“antagomirs”. An antagomir is an RNA oligonucleotide or RNAoligonucleotide mimetic having complementarity to a specific miRNA, andwhich inhibits the activity of that miRNA.

In some embodiments, the inhibitor of a transcription factor repressoris an antagomir targeting one or more of the miRNAs listed in Table 3.The antagomir may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotidedifferences from the complementary sequence of the miRNA that itinhibits. Further, antagomirs may have the same length, a longer length,or a shorter length than the miRNA that they inhibit. In certainembodiments, the antagomir hybridizes to 6-8 nucleotides at the 5′ endof the miRNA it inhibits. In other embodiments, the antagomir iscomplementary to a miRNA is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotidesin length. In other embodiments, the antagomir is 5-10, 6-8, 10-20, 10-15 or 5-500 nucleotides in length.

In some embodiments, the antagomir is a synthetic reverse complement ofa target RNA that tightly binds to and inactivates the target miRNA.Thus, in some embodiments, the antagomir specifically inhibits or bocksthe expression or activity of the target miRNA.

In some embodiments, the antagomir is at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% complimentary to any oneof the miRNA sequence provided in Table 3 above.

In some embodiments, the antagomir is capable of inhibiting theexpression of one or more miRNAs disclosed in Table 3.

In particular, the antagomirs of the present application may besubstantially complementary to nucleic acid sequence specific to miRNAselected from miR-193a-5p, miR-23b-3p, miR-4687-3p, miR-4651, miR-4270,or miR-24-3p, or to a portion thereof. Accordingly, nucleic acids oranalogs thereof displaying substantially equivalent or altered activityare likewise contemplated.

In some embodiments, the antagomir comprises chemical modificationswhich improve nuclease resistance and binding affinity. Suitablemodifications include, without limitation, 2′ sugar modifications, suchas 2′-O-Me, 2′-O-methoxyethyl (2′-MOE), or 2′-fluoro(2′-F).

In some embodiments, the expressing step in the method of inducingrejuvenation in adult GPCs , or in the method of treating myelindeficiency in a subject, comprises administering to the adult GPCs orGPCs in the subject, respectively, an effective amount of (1) aninhibitor of a transcription factor repressor, and/or (2) an effectiveamount of one or more transcription factors selected from the groupconsisting of B-cell lymphoma/leukemia 11A (BCL11A), histone deacetylase2 (HDAC2), histone-lysine N-methyltransferase EZH2 (EZH2), mycproto-oncogene protein (MYC), high 15 mobility group protein HMGI-C(HMGA2), nuclear factor 1 B-type (NFIB) and transcriptional enhancerfactor TEF-4 (TEAD2).

In some embodiments, the expressing step in the method of inducingrejuvenation in adult GPCs , or in the method of treating myelindeficiency in a subject, comprises administering to the adult GPCs orGPCs in the subject, respectively, an effective amount of (1) anexpression vector encoding an inhibitor of a transcription factorrepressor, and/or (2) an effective amount of an expression vectorencoding one or more transcription factors selected from the groupconsisting of B-cell lymphoma/leukemia 11A (BCL11A), histone deacetylase2 (HDAC2), histone-lysine N-methyltransferase EZH2 (EZH2), mycproto-oncogene protein (MYC), high 15 mobility group protein HMGI-C(HMGA2), nuclear factor 1 B-type (NFIB) and transcriptional enhancerfactor TEF-4 (TEAD2).

In some embodiments, the regulatory sequence of the expression vectordescribed above in the method of inducing rejuvenation in adult GPCs, orin the method of treating myelin deficiency in a subject, comprises apromoter and/or enhancer for a gene selectively or specificallyexpressed by glial progenitor cells.

Genes selectively expressed by glial progenitor cells include platelet25 derived growth factor alpha (PDGFRA), zinc finger protein 488(ZNF488), G protein-coupled receptor (GPR17), oligodendrocyteTranscription Factor 2 (OLIG2), chondroitin sulfate proteoglycan 4(CSPG4), and SRY-box transcription factor 10 (SOX10).

In accordance with such embodiments, promoter sequences suitable forcontrolling expression of the nucleic acid inhibitors disclosed hereininclude, without limitation, the platelet derived growth factor alpha(PDGFRA) promoter, the zinc finger protein 488 (ZNF488) promoter, the Gprotein-coupled receptor (GPR17) promoter, the oligodendrocyteTranscription Factor 2 (OLIG2) promoter, the chondroitin sulfateproteoglycan 4 (CSPG4) promoter, and the SRY-box transcription factor 10(SOX10) promoter, which are identified in Table 4 below.

TABLE 4 Exemplary Genes which are Selectively or Specifically Expressedby Glial Progenitor Cells Gene NCBI ID Reference Gene Organism No.*Sequence:* Platelet derived Homo sapiens 5156 NG_009250.1 growth factoralpha (PDGFRA) Zinc finger protein Homo sapiens 118738 488 (ZNF488) Gprotein-coupled Homo sapiens 2840 NG_042235.1 receptor (GPR17)Oligodendrocyte Homo sapiens 10215 NG_011834.1 Transcription Factor 2OLIG2) chondroitin sulfate Homo sapiens 1464 proteoglycan 4 (CSPG4)SRY-box transcription Homo sapiens 6663 NG_007948.1 factor 10 (SOX10)*Each of which is hereby incorporated by reference in its entirety.

In some embodiments, the regulatory sequence of the expression vectordescribed above in the method of inducing rejuvenation in adult GPCs, orin the method of treating myelin deficiency in a subject, comprises aninducible promoter or promoter system, such as tetracycline controlledinducible system, cumate-controlled inducible system and rapamycincontrolled inducible systems, which are described in more detail infra.

In some embodiments, the expression vector described above in the methodof inducing rejuvenation in adult GPCs, or in the method of treatingmyelin deficiency in a subject, is a plasmid vector, a viral vector, ora bacterial vector.

In some embodiments, the expression vector described above in the methodof inducing rejuvenation in adult GPCs, or in the method of treatingmyelin deficiency in a subject, is a viral vector selected from thegroup consisting of adenoviruses, adeno-associated viruses (AAVs),retroviruses, lentiviruses, vaccinia viruses, and herpes viruses. Insome embodiments, the expression vector is a lentiviral vector. In someembodiments, the expression vector is a retroviral vector. In otherembodiments, the expression vector is an AAV vector. Methods forgenerating and isolating viral vectors suitable for use as expressionvectors are described in more details infra.

III. Methods Involving the Suppression of Trascription Factors

Another aspect of the present application relates to a method ofinducing rejuvenation in a population of adult glial progenitor cells bysuppressing certain transcription factors. The method involvesadministering, to the population of adult glial progenitor cells, aneffective amount of an agent that suppresses one or more transcriptionfactor selected from the group consisting of zinc finger protein 274(ZNF274), Myc-associated factor X (MAX), E2F transcription factor 6(E2F6), zinc finger protein Aiolos (IKZF3), and signal transducer andactivator of transcription 3 (STAT3).

Another aspect of the present application relates to a method ofinducing rejuvenation in a population of adult glial progenitor cells bysuppressing certain senescence genes. The method involves administering,to the population of adult glial progenitor cells, an effective amountof an agent that inhibits expression of one or more senescence genesselected from the group consisting of RUNX1, BIN1, VAMP3, DMTF1, CTNNA1,SERPINEL CDK19, CDKN1C, RUNX2, EFEMP1, MAP3K7, AHR, OGT, PAK1, CBX7, andCYLD.

Another aspect of the present application relates to a method ofinducing rejuvenation in a population of adult glial progenitor cells bysuppressing certain senescence genes. The method involves administering,to the population of adult glial progenitor cells, an effective amountof an agent that inhibits expression of one or more glial target genesselected from the group consisting of MBP, CD9, ENPP2, PLP1, ERBIN,ZNF365, UGT8, GDNF, DUSP10, PMP22, ERBB4, and MYRF.

Adult glial progenitor cells suitable for use in the methods disclosedherein include mammalian glial progenitor cells, e.g., human glialprogenitor cells, rodent glial progenitor cells, non-human primate glialprogenitor cells, ovine glial progenitor cells, bovine glial progenitorcells, porcine glial progenitor cells, canine glial progenitor cells,and feline glial progenitor cells. In some embodiments, the adult glialprogenitor cells are adult human glial progenitor cells.

In some embodiments, said administering is carried out ex vivo. In otherembodiments of the methods according to the present disclosure, saidadministering is carried out in vivo.

Another aspect of the present application relates to a method oftreating a myelin deficiency in a subject by suppressing certaintranscription factors. This method involves administering, to thesubject having the myelin deficiency, an agent that suppresses one ormore transcription factors selected from the group consisting of ZNF274,MAX, E2F6, IKZF3, and STAT3, wherein the agent is administered in aneffective amount to suppress the one or more transcription factors inadult glial progenitor cells of the subject.

Another aspect of the present application relates to a method oftreating a myelin deficiency in a subject by suppressing certainsenescence genes. The method involves administering, to the subject, anagent that inhibits expression of one or more senescence genes selectedfrom the group consisting of RUNX1, BIN1, VAMP3, DMTF1, CTNNA1, SERPINELCDK19, CDKN1C, RUNX2, EFEMP1, MAP3K7, AHR, OGT, PAK1, CBX7, and CYLD,wherein the agent is administered in an effective amount to suppress theone or more senescence genes in adult glial progenitor cells of thesubject.

Another aspect of the present application relates to a method oftreating a myelin deficiency in a subject by suppressing certain glialtarget genes. The method involves administering, to the subject, anagent that inhibits expression of one or more senescence genes selectedfrom the group consisting of MBP, CD9, ENPP2, PLP1, ERBIN, ZNF365, UGT8,GDNF, DUSP10, PMP22, ERBB4, and MYRF, wherein the agent is administeredin an effective amount to suppress the one or more glial target genes inadult glial progenitor cells of the subject.

In accordance with this aspect of the present application, the myelindeficiency may be associated with a condition selected from the groupconsisting of multiple sclerosis, neuromyelitis optica, transversemyelitis, optic neuritis, subcortical stroke, diabeticleukoencephalopathy, hypertensive leukoencephalopathy, age-related whitematter disease, spinal cord injury, radiation- or chemotherapy induceddemyelination, post-infectious and post-vaccinial leukoencephalitis,periventricular leukomalacia, pediatric leukodystrophy (e.g.,Pelizaeus-Merzbacher Disease, Tay-Sach Disease, Sandhoff sgangliosidoses, Krabbe's disease, metachromatic leukodystrophy,mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy,Canavan's disease, Vanishing White Matter Disease, and AlexanderDisease), lysosomal storage diseases, congenital dysmyelination,inflammatory demyelination, vascular demyelination, and cerebral palsy.

In some embodiments, the myelin deficiency is associated with aneurodegenerative disease, e.g., Huntington's disease. As used herein,“Huntington's disease” refers to an autosomal dominant inherited braindisorder that typically becomes manifest in adulthood. Huntington'sdisease pathology is characterized by hypomyelination, as well asneuronal and white matter loss.

In other embodiments, the myelin deficiency is associated with aneuropsychiatric disease, e.g., schizophrenia.

In some embodiments, the agent used in the method of inducingrejuvenation or the method of treating myelin deficiency is an agentthat suppresses STAT3. As illustrated in FIG. 4F of the presentdisclosure, in the context of adult glial progenitor cells, STAT3 ispredicted to activate a set of senescence-associated genes (e.g., BIN1,DMTF1, CD47, CTNNA1, RUNX2, RUNX1, MAP3K7, and OGT), glialcell-associated genes (e.g., PLP1, CNP, PMP22, SEMA4D, CLDN11, GPR37,MYRF, MAG, BCAS1, ST18, ERBB4, CERS2, LPAR1, and GJB1), and downstreamtranscription factors (e.g., MAX, E2F6, and IKZF3). In some embodiments,the agent is an agent that suppresses or silences the activity of STAT3and/or any of the senescence- associated genes noted above that areactivated by STAT3.

In some embodiments, the agent used in the method inducing rejuvenationand the method of treating myelin deficiency is an agent that suppressesone or more of ZNF274, MAX, E2F6, and IKZF3. As illustrated in FIG. 4Gof the present application, in the context of adult glial progenitorcells, ZNF274, MAX, E2F6, and IKZF3 are predicted to repress sets ofproliferation-associated gene targets (e.g., YAP1, LMNB1, PATZ1, TEAD1,FN1, TP53, CDK1, CCND2, CDKN2D, CENPH, MKI67, CDK4, CENPF, CDK5, CDKN3,and CHEK1), glial cell-associated genes (e.g., CHRDL1, ST8SIA1, PTPRZ1,CA10, PDGFRA, BCAN, NXPH1, CSPG4, and, PCDH15), and downstreamtranscription factors (e.g., BLC11A, EZH2, HDAC2, NF1B, MYC, HMGA2, andTEAD).

In some embodiments, the agent is an agent that suppresses or silencesthe activity of ZNF274, MAX, E2F6, IKZF3, or any combination thereof toallow the expression of downstream proliferation-associated genetargets.

In some embodiments, the agent is an agent that suppresses ZNF274, MAX,E2F6, IKZF3, and/or STAT3. Suitable agents for use in the methodsdescribed herein include, without limitation, a ZNF274 inhibitor, a MAXinhibitor, an E2F6 inhibitor, an IKZF3 inhibitor, and a STAT3 inhibitor.

The ZNF274 inhibitor, MAX inhibitor, E2F6 inhibitor, IKZF3 inhibitor,and/or STAT3 inhibitor may be (1) a small molecule inhibitor, (2) anucleic acid molecule inhibitor (e.g., a miRNA, a shRNAi, a siRNA, andan antisense oligonucleotide), (3) a nuclease-based gene editing system(e.g., a CRISPR/Cas-system targeted to silence ZNF274, MAX, E2F6, IKZF3,and/or STAT expression), or (4) a nucleic acid molecule encoding (2) or(3).

Small Molecule Inhibitors

In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6inhibitor, IKZF3 inhibitor, and/or STAT3 inhibitor is a small moleculeinhibitor. Exemplary small molecule inhibitors of these transcriptionfactors that are known in the art and suitable for use in accordancewith the methods described herein are provided in Table 5 below. Analogsand derivatives of the small molecule inhibitors of Table 5 are alsocontemplated for use in the methods described herein.

TABLE 5 Exemplary Small Molecule Inhibitors Transcription FactorExemplary Small Molecule Inhibitors MYC-associated factor X (MAX)inhibitor

  100-58-F4 (1RH)

  12RH

  22RH

  27RH

  28RH

  1RH-S-Me

  1RH-NCN-1

  #015

  #474

  #764

  12RH-NCN-1

  28RH-NCN-1

  MYCMI-6

  MYCMI-11

  MYCMI-14 Zinc finger protein Aiolos (IKZF3)

  Thalidomide

  Lenalidomide

  Pomalidomide

  CC-122

  CC-885

  CC-220 Signal transducer and activator of transcription 3 (STAT3)

  Cryptotanshinone

  STA-21

  NSC 74859 (S31-201)

  Napabucasin

  Stattic, 1

  Cucurbitacin I JSI-124

  Cucurbitacin Q

  Phpr-pTyr-Leu-cis-3,4-methanoPro-Gln-NHBn

  LLL-3

  LLL-12

  S31-201

  SF-1-066

  S31-1757

  STX-0119

  Cpd30-12

  LY5

  Cpd9

  Cpd1

In some embodiments, the small molecule inhibitor is a small moleculeinhibitor of Myc-associated factor X (MAX) selected from the groupconsisting of 10058-F4 (also known as “10058-F4 (1RH)”) (see, e.g.,Huang et al., “A Small-Molecule c-Myc Inhibitor, 10058-F4, InducesCell-Cycle Arrest, Apoptosis, and Myeloid Differentiation of Human AcuteMyeloid Leukemia,” Exp. Hematol. 34(11):1480-1489 (2006), which ishereby incorporated by reference in its entirety); MYCMI-6, MYCMI-11,and MYCMI-14 (see, e.g., Castell et al., “A Selective High AffinityMYC-Binding Compound Inhibits MYC:MAX Interaction and MYC-DependentTumor Cell Proliferation,” Sci. Rep. 8:10064 (2018), which is herebyincorporated by reference in its entirety); 12RH, 22RH, 27RH, 28RH,1RH-S-Me, 1RH-NCN-1, #015, #474, #764, 12RH-NCN-1, and 28RH-NCN-1 (see,e.g., Wang et al., “Improved Low Molecular Weight Myc-Max inhibitors,”Mol. Cancer Ther. 6(9):2399-2408 (2007), which is hereby incorporated byreference in its entirety).

In some embodiments, the small molecule inhibitor is a small moleculeinhibitor of signal transducer and activator of transcription 3 (STAT3)selected from the group consisting of cryptotanshinone (see, e.g., Shinet al., “Cryptotanshinone Inhibits Constitutive Signal Transducer andActivator of Transcription 3 Function through Blocking the Dimerizationin DU145 Prostate Cancer Cells,” Cancer Res. 69(1):193-202 (2009), whichis hereby incorporated by reference in its entirety); STA-21 (see, e.g.,Song et al., “A Low-Molecular-Weight Compound Discovered through VirtualDatabase Screening Inhibits Stat3 Function in Breast Cancer Cells,” PNAS102(13):4700-4705 (2005), which is hereby incorporated by reference inits entirety); NSC 7459 (S3I-201) (see, e.g., Siddiquee et al.,“Selective Chemical Probe Inhibitor of Stat3, Identified throughStructure-Based Virtual Screening, Induces Antitumor Activity,” PNAS104(18):7391-7396 (2007), which is hereby incorporated by reference inits entirety); napabucasin (BBI608) (see, e.g., Li et al., “Suppressionof Cancer Relapse and Metastasis by Inhibiting Cancer Stemness,” PNAS112(6):1839-1844 (2015) and Hubbard et al., “Napabucasin: An Update onthe First-in-Class Cancer Stemness Inhibitor,” Drugs 77(10):1091-1103(2017), which are hereby incorporated by reference in their entirety);Stattic, cucurbitacin I, cucurbitacin Q,Phpr-pTyr-Leu-cis-3,4-methanoPro-Gln-NHBn (see, e.g., McMurray, J., “ANew Small-Molecule Stat3 Inhibitor,” Chem. Biol. 13(11):1123-1124(2006), which is hereby incorporated by reference in its entirety);LLL-3 (see, e.g., Fuh et al., “LLL-3 Inhibits STAT3 Activity, SuppressesGlioblastoma Cell Growth and Prolongs Survival in a Mouse GlioblastomaModel,” Br. J. Cancer 100(1):106-112 (2009), which is herebyincorporated by reference in its entirety); LLL- 12, S31-201, SF-1-066,S31-1757, STX-0119, Cpd30-12, LYS, Cpd9, and Cpd1 (see, e.g., Orlova etal., “Direct Targeting Options for STAT3 and STATS in Cancer,” Cancers11(12):1930 (2019), which is hereby incorporate by reference in itsentirety); SF-1-087, SF-1-121, S3I-M2001, S3I-201.1066, and BP-1-102(see, e.g., Wu et al., “Negative Regulators of STAT Signaling Pathway inCancers,” Cancer Manag. Res. 11:4957-4969 (2019), which is herebyincorporated by reference in its entirety); HO-3867 (see, e.g., Tierneyet al., “HO-3867, a STAT3 Inhibitor Induces Apoptosis by Inactivation ofSTAT3 Activity in BRCA1-Mutated Ovarian Cancer Cells,” Cancer Biol.Ther. 13(9):766-775 (2012); corylifol A (see, e.g., Lee et al.,“Phenolic Compounds Isolated from Psoralea corylifolia InhibitIL-6-Induced STAT3 Activation,” Planta. Med. 78(9):903-906 (2012), whichis hereby incorporated by reference in its entirety); and SD 1008 (see,e.g., Liu et al., “SOCS3 Promotes Inflammation and Apoptosis viaInhibiting JAK2/STAT3 Signaling Pathway in 3T3-L1 Adipocyte,”Immunobiology 220(8):947-953 (2015), which is hereby incorporated byreference in its entirety).

Nucleic Acid Inhibitors

In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6inhibitor, IKZF3 inhibitor, and/or STAT3 inhibitor is a nucleic acidmolecule inhibitor.

As used herein, the term “nucleic acid molecule inhibitor” refers to anucleic acid molecule that reduces or eliminates the expression of atarget gene. The nucleic acid molecule inhibitor typically contains aregion that specifically targets a sequence in the target gene or targetgene mRNA to achieve target-specific inhibition. Typically, thetargeting region of the nucleic acid inhibitor molecule comprises asequence that is sufficiently complementary to a sequence on the targetgene or target gene mRNA to direct the effect of the nucleic acidinhibitor molecule to the specified target gene or target gene mRNA. Forexample, a “nucleic acid molecule inhibitor of ZNF274” reduces oreliminates the expression of a ZNF274 gene. The nucleic acid inhibitormolecule may include natural ribonucleotides, naturaldeoxyribo-nucleotides, and/or modified nucleotides. The modifiednucleotides include modifications such as substitution on positions onthe sugar ring, modifications of the phosphoester linkages betweennucleotides, non-natural bases, and non-natural alternative carbonstructures such as locked nucleic acids (“LNA”) and unlocked nucleicacids (“UNA”).

As used herein, the term “reduce” or “reduces” refers to its meaning asis generally accepted in the art. With reference to exemplary nucleicacid inhibitor molecules (e.g., nucleic acid molecule inhibitors ofZNF274, MAX, E2F6, IKZF3, and/or STAT3 selected from a miRNA, a shRNAi,a siRNA, and an antisense oligonucleotide), reduce” or “reduces”generally refers to a suppression in the transcription and/ortranslation of a gene or in the levels of the gene product relative tothe transcription and/or translation of the gene observed in the absenceof the nucleic acid inhibitor molecule. In some embodiments, thereduction in the transcription and/or translation of a gene or in thelevels of the gene product is at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, up to 100% (i.e., no detectable transcription and/ortranslation) or a reduction of at least 2-fold, at least 5-fold, atleast 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, atleast 50-fold, or more relative to that observed in the absence of thenucleic acid inhibitor molecule according to the present disclosure.

Suitable nucleic acid inhibitor molecules include, without limitation,(i) a nucleic acid molecule inhibitor of ZNF274 selected from a miRNA, ashRNAi, a siRNA, and an antisense oligonucleotide (ASO); (ii) a nucleicacid molecule inhibitor of MAX selected from a miRNA, a shRNAi, a siRNA,and an antisense oligonucleotide; (iii) a nucleic acid moleculeinhibitor of E2F6 selected from a miRNA, a shRNAi, a siRNA, and anantisense oligonucleotide; (iv) a nucleic acid molecule inhibitor ofIKZF3 selected from a miRNA, a shRNAi, a siRNA, and an antisenseoligonucleotide; and (v) a nucleic acid molecule inhibitor of STAT3selected from a miRNA, a miRNA inhibitor, a shRNAi, a siRNA, and anantisense oligonucleotide.

As use herein, the term “microRNA” or “miRNA” refers to a class of smallRNA molecules that may negatively regulate gene expression (see, e.g.,Lam et al., “siRNA Versus miRNA as Therapeutics for Gene Silencing,”Mol. Ther. Nucleic Acids 4(9):e252 (2015), which is hereby incorporatedby reference in its entirety). miRNA gene transcription is carried outby RNA polymerase II in the nucleus to give primary miRNA (pri-miRNA),which is a 5′ capped, 3′ polyadenylated RNA with double-strandedstem-loop structure. The pri-miRNA is then cleaved by a microprocessorcomplex (comprising Drosha and microprocessor complex subunit DCGR8) toform precursor miRNA (pre-miRNA), which is a duplex that contains 70-100nucleotides with interspersed mismatches and adopts a loop structure.The pre-miRNA is subsequently transported by Exportin 5 from the nucleusto the cytoplasm, where it is further processed by Dicer into a miRNAduplex of 18-25 nucleotides. The miRNA duplex then associates with theRISC forming a complex called miRISC. The miRNA duplex is unwound,releasing and discarding the passenger strand (sense strand). The maturesingle-stranded miRNA guides the miRISC to the target mRNAs. MaturemiRNA may bind to a target mRNA through partial complementary basepairing with the consequence that the target gene silencing occurs viatranslational repression, degradation, and/or cleavage. In someembodiments, the nucleic acid inhibitor molecule is a miRNA molecule.Suitable miRNA inhibitor molecules for use in the methods of the presentdisclosure include, without limitation, those identified in Table 6below.

TABLE 6 Exemplary miRNA sequences Trans- SEQ cription ID  Factor miRNASequence NO: MAX miR-485-5p AGAGGCUGGCCGUGAUGAAUUC  7 E2F6 miR-379-5pUGGUAGACUAUGGAACGUAGG  8 STAT3 miR-125b-5p UCCCUGAGACCCUAACUUGUGA  9STAT3 miR-106a-5p AAAAGUGCUUACAGUGCAGGUAG 10 STAT3 miR-17-5pCAAAGUGCUUACAGUGCAGGUAG 11 STAT3 miR-130a-3p CAGUGCAAUGUUAAAAGGGCAU 12STAT3 miR-130b-3p CAGUGCAAUGAUGAAAGGGCAU 13

In some embodiments, the MAX inhibitor is a nucleic acid moleculeinhibitor of MAX, and a suitable MAX nucleic acid molecule inhibitor isa miRNA. In accordance with such embodiment, exemplary MAX inhibitormiRNAs have a nucleotide sequence corresponding to miR-485-5p,pre-miR-485-5p, or mature miR-485-5p. For example, the miRNA may havethe nucleotide sequence of SEQ ID NO:7.

In some embodiments, the E2F6 inhibitor is a nucleic acid moleculeinhibitor of E2F6, and a suitable E2F6 nucleic acid molecule inhibitoris a miRNA. In accordance with such embodiment, exemplary E2F6 inhibitormiRNAs have a nucleotide sequence corresponding to miR-379-5p,pre-miR-379-5p, or mature miR-379-5p. For example, the miRNA may havethe nucleotide sequence of SEQ ID NO:8.

In some embodiments, the STAT3 inhibitor is a nucleic acid moleculeinhibitor of STAT3, and a suitable STAT3 inhibitor is a miRNA. Inaccordance with such embodiment, exemplary STAT3 inhibitory miRNAs havea nucleotide sequence corresponding to miR-125b- 5p, pre-miR-125b-5p,mature miR-125b-5p, miR-106a-5p, pre-miR-106a-5p, mature miR-106a-5p,miR-17-5p, pre-miR-17-5p, mature miR-17-5p, miR-130a-3p,pre-miR-130a-3p, mature miR-130a-3p, miR-130b-3p, pre-miR-130b-3p, ormature miR-130b-3p. For example, the miRNA may have the nucleotidesequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, orSEQ ID NO:13.

In some embodiments, the agent used in the method of inducingrejuvenation and the method of treating myelin deficiency comprises oneor more expression vectors encoding (i) one or more microRNA selectedfrom the group consisting of miR-125b-5p, miR-106a-5p, miR-17-5p,miR-130a-3p, and miR-130b-3p, wherein said administering suppresses thesignal transducer and activator of transcription 3 (STAT3) signalingpathway; (ii) miR-379-5p, wherein said administering suppresses the E2Ftranscription factor 6 (E2F6) signaling pathway; and/or (iii)miR-485-5p, wherein said administering suppresses the Myc-associatedfactor X (MAX) signaling pathway.

In some embodiments, the agent used in the method of inducingrejuvenation and the method of treating myelin deficiency comprises anexpression vectors encoding an MAX inhibitor. In some embodiments, theMAX inhibitor is a nucleic acid molecule inhibitor. In some embodiments,the MAX nucleic acid molecule inhibitor is a miRNA. In accordance withsuch an embodiment, exemplary MAX inhibitor miRNAs have a nucleotidesequence corresponding to miR-485-5p, pre-miR-485-5p, or maturemiR-485-5p. For example, the miRNA may have the nucleotide sequence ofSEQ ID NO:7.

In some embodiments, the agent used in the method of inducingrejuvenation and the method of treating myelin deficiency comprises anexpression vector encoding an E2F6 inhibitor. In some embodiments, theE2F6 inhibitor is a nucleic acid molecule inhibitor. In someembodiments, the E2F6 nucleic acid molecule inhibitor is a miRNA. Inaccordance with such embodiment, exemplary E2F6 inhibitor miRNAs have anucleotide sequence corresponding to miR-379-5p, pre- miR-379-5p, ormature miR-379-5p. For example, the miRNA may have the nucleotidesequence of SEQ ID NO:8.

In some embodiments, the agent used in the method of inducingrejuvenation and the method of treating myelin deficiency comprises anexpression vector encoding an STAT3 inhibitor. In some embodiments, theSTAT3 inhibitor is a nucleic acid molecule inhibitor. In someembodiments, the STAT3 nucleic acid molecule inhibitor is a miRNA. Inaccordance with such embodiment, exemplary STAT3 inhibitory miRNAs havea nucleotide sequence corresponding to miR-125b-5p, pre-miR-125b-5p,mature miR- 125b-5p, miR-106a-5p, pre-miR-106a-5p, mature miR-106a-5p,miR-1′7-5p, pre-miR 5p, mature miR-17-5p, miR-130a-3p, pre-miR-130a-3p,mature miR-130a-3p, miR-130b-3p, pre-miR-130b-3p, or mature miR-130b-3p.For example, the miRNA may have the nucleotide sequence of SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

In some embodiments, the agent used in the method of inducingrejuvenation and the method of treating myelin deficiency comprises anexpression vector that encodes (1) one or more microRNA selected fromthe group consisting of miR-485-5p miR-379-5p, miR-125b-5p, miR-106a-5p,miR-17-5p, miR-130a-3p and miR-130b-3p, and (2) one or more microRNAselected from the group consisting of miR-93-3p, miR-1260b, miR-767-5p,miR-30b-5p, miR-9-3p, and miR-9-5p, as shown in Table 7 below.

TABLE 7 miRNA Sequences miRNA Sequence SEQ ID NO: miR-93-3pACUGCUGAGCUAGCACUUCCCG  8 miR-1260b AUCCCACCACUGCCACCAU  9 miR-767-5pUGCACCAUGGUUGUCUGAGCAUG 10 miR-30b-5p UGUAAACAUCCUACACUCAGCU 11 miR-9-3pAUAAAGCUAGAUAACCGAAAGU 12 miR-9-5p UCUUUGGUUAUCUAGCUGUAUGA 13

In some embodiments, the nucleic acid inhibitor molecule of ZNF274, MAX,E2F6, IKZF3, and/or STAT3 is an shRNA molecule. Short hairpin RNA(shRNA) molecules comprise the sense and antisense sequences from atarget gene connected by a loop. Once transcribed, shRNA molecules aretransported from the nucleus into the cytoplasm where the enzyme Dicerprocesses them into small/short interfering RNAs (siRNAs) in a shorthairpin RNA interference process. As used herein, the term “shorthairpin RNA interference” or “shRNAi” is a process mediated by a classof small RNA molecules that negatively regulate gene expression.

In some embodiments, the ZNF274 inhibitor is a nucleic acid moleculeinhibitor of ZNF274, and a suitable ZNF274 inhibitor is a ZNF274 shRNA.In some embodiments, the MAX inhibitor is a nucleic acid moleculeinhibitor of MAX, and a suitable MAX inhibitor is a MAX shRNA. In someembodiments, the E2F6 inhibitor is a nucleic acid molecule inhibitor ofE2F6, and a suitable E2F6 inhibitor is an E2F6 shRNA. In someembodiments, the IKZF3 inhibitor is a nucleic acid molecule inhibitor ofIKZF3, and a suitable IKZF3 inhibitor is an IKZF3 shRNA. In someembodiments, the STAT3 inhibitor is a nucleic acid molecule inhibitor ofSTAT3, and a suitable STAT3 inhibitor is a STAT3 shRNA.

In some embodiments, the nucleic acid inhibitor molecule of ZNF274, MAX,E2F6, IKZF3, and/or STAT3 is a siRNA. As used herein, the term “shortinterfering RNA” or “siRNA” refers to short nucleic acid moleculestypically 21-23 nucleotides in length with 3′-two nucleotide overhangs(see, e.g., McManus & Sharp, “Gene Silencing in Mammals by SmallInterfering RNAs,” Nat. Rev. Genet. 3(10):737-747 (2002), which ishereby incorporated by reference in its entirety). siRNA interacts withand activates the RNA-induced silencing complex (“RISC”). Theendonuclease argonaute 2 (AGO2) component of the RISC cleaves thepassenger strand (sense strand) of the siRNA while the guide strand(antisense strand) remains associated with the RISC.

Subsequently, the guide strand guides the active RISC to its target mRNAfor cleavage by AGO2. As the guide strand only binds to mRNA that isfully complementary to it, siRNA causes specific gene silencing (see,e.g., Lam et al., “siRNA Versus miRNA as Therapeutics for GeneSilencing,” Mol. Ther. Nucleic Acids 4(9):e252 (2015), which is herebyincorporated by reference in its entirety).

In some embodiments, the ZNF274 inhibitor is a ZNF274 siRNA. In someembodiments, the MAX inhibitor is a MAX siRNA. In some embodiments, theE2F6 inhibitor is an E2F6 siRNA. In some embodiments, the IKZF3inhibitor is an IKZF3 siRNA. In some embodiments, the STAT3 inhibitor isa STAT3 siRNA.

In some embodiments, the nucleic acid inhibitor molecule of ZNF274, MAX,E2F6, IKZF3, and/or STAT3 is an antisense oligonucleotide. As usedherein, the term “antisense oligonucleotide” or “ASO” refers to small(-18-30 nucleotides), synthetic, single-stranded nucleic acid polymersof diverse chemistries, which can be employed to modulate geneexpression via various mechanisms (see, e.g., Roberts et al., “Advancesin Oligonucleotide Drug Delivery,” Nature Reviews Drug Discovery19:673-694 (2020), which is hereby incorporated by reference in itsentirety). ASOs can be subdivided into two major categories: RNase Hcompetent and steric block. The endogenous RNase H enzyme RNASEH1recognizes RNA—DNA heteroduplex substrates that are formed whenDNA-based oligonucleotides bind to their cognate mRNA transcripts andcatalyzes the degradation of RNA. Cleavage at the site of ASO bindingresults in destruction of the target RNA, thereby silencing target geneexpression. Steric block oligonucleotides are ASOs that are designed tobind to target transcripts with high affinity but do not induce targettranscript degradation as they lack RNase H competence. Sucholigonucleotides therefore comprise either nucleotides that do not formRNase H substrates when paired with RNA or a mixture of nucleotidechemistries (that is, ‘mixmers’) such that runs of consecutive DNA-likebases are avoided.

In some embodiments, the ZNF274 inhibitor is a ZNF274 ASO. In someembodiments, the MAX inhibitor is a MAX ASO. In some embodiments, theE2F6 inhibitor is an E2F6 ASO. In some embodiments, the IKZF3 inhibitoris an IKZF3 ASO. In some embodiments, the STAT3 inhibitor is a STAT3ASO, e.g., danvatirsen (see, e.g., Xu et al., “PopulationPharmacokinetic Analysis of Danvatirsen Supporting Flat Dosing Switch,”J. Pharmacokinet. Pharmacodyn. 46(1):65-74 (2019), which is herebyincorporated by reference in its entirety).

Methods of designing nucleic acid inhibitors are well known in the artand suitable for designing nucleic acid inhibitors for use in themethods described herein (see, e.g., Lam et al., “siRNA Versus miRNA asTherapeutics for Gene Silencing,” Mol. Ther. Nucleic Acids 4(9):e252(2015) and Kulkarni et al., “The Current Landscape of Nucleic AcidTherapeutics,” Nature Nanotechnology 16:630-643 (2021), which are herebyincorporated by reference in their entirety).

Nucleic acid inhibitor molecules are designed to target ZNF274, MAX,E2F6, IKZF3, and/or STAT3 and transcription variants thereof in asequence specific manner. The sequences of ZNF274, MAX, E2F6, IKZF3,and/or STAT3 and transcription variants thereof are well known in theart and accessible via various curated databases, e.g., NCBI nucleotideor gene database. In some embodiments, the nucleic acid inhibitormolecule is designed to target one or more of the transcription factorsidentified in Table 8 below using the sequences available via the NCBIAccession number provided.

TABLE 8 Exemplary Human Transcription Factor Genes and TranscriptVariants Gene Reference Transcription ID Transcript Transcript FactorNo.* Variant Accession Nos.* zinc finger 10782 ZNF274c NM_133502.3protein 274 ZNF274b NM_016324.4 (ZNF274) ZNF274a NM_016325.4 ZNF274dNM_001278734.2 transcript variant X1 XM_011526327.1 transcript variantX3 XM_011526328.2 transcript variant X2 XM_017026174.1 transcriptvariant X5 XM_017026175.1 transcript variant X4 XR_001753588.2transcript variant X6 XR_001753589.2 Myc-associated 4149 transcriptvariant 1 NM_002382.5 factor X (MAX) transcript variant 2 NM_145112.3transcript variant 3 NM_145113.3 transcript variant 4 NM_145114.3transcript variant 6 NM_197957.4 transcript variant 7 NM_001271068.2transcript variant 8 NM_001271069.2 transcript variant 11 NM_001320415.2transcript variant 9 NR_073137.1 transcript variant 10 NR_073138.1transcript variant X1 XM_011536773.3 transcript variant X7XM_017021312.2 transcript variant X8 XM_017021313.1 transcript variantX3 XR_943450.3 transcript variant X4 XR_943451.3 transcript variant X5XR_943452.3 transcript variant X6 XR_001750326.2 transcript variant X9XR_001750327.2 transcript variant X2 XR_002957553.1 E2F 1876 transcriptvariant a NM_198256.4 transcription transcript variant f NM_212540.3factor 6 (E2F6) transcript variant b NM_001278275.2 transcript variant cNM_001278276.2 transcript variant d NM_001278277.2 transcript variant eNM_001278278.2 transcript variant g NR_103490.2 transcript variant X1XM_017003547.1 transcript variant X2 XM_017003548.1 transcript variantX3 XM_017003549.2 transcript variant X4 XR_001738660.1 zinc finger 22806transcript variant 1 NM_012481.5 protein Aiolos transcript variant 2NM_183228.3 (IKZF3) transcript variant 3 NM_183229.3 transcript variant4 NM_183230.3 transcript variant 5 NM_183231.3 transcript variant 6NM_183232.3 transcript variant 7 NM_001257408.2 transcript variant 8NM_001257409.2 transcript variant 9 NM_001257410.2 transcript variant 10NM_001257411.2 transcript variant 11 NM_001257412.2 transcript variant12 NM_001257413.2 transcript variant 13 NM_001257414.2 transcriptvariant 14 NM_001284514.2 transcript variant 15 NM_001284515.2transcript variant 16 NM_001284516.1 signal transducer 6774 transcriptvariant 1 NM_139276.3 and activator of transcript variant 2 NM_003150.4transcription 3 transcript variant 3 NM_213662.2 (STAT3) transcriptvariant 4 NM_001369512.1 transcript variant 5 NM_001369513.1 transcriptvariant 6 NM_001369514.1 transcript variant 7 NM_001369516.1 transcriptvariant 8 NM_001369517.1 transcript variant 9 NM_001369518.1 transcriptvariant 10 NM_001369519.1 transcript variant 11 NM_001369520.1transcript variant 12 NM_001384984.1 transcript variant 13NM_001384985.1 transcript variant 14 NM_001384986.1 transcript variant15 NM_001384987.1 transcript variant 16 NM_001384988.1 transcriptvariant 17 NM_001384989.1 transcript variant 18 NM_001384990.1transcript variant 19 NM_001384991.1 transcript variant 20NM_001384992.1 transcript variant 21 NM_001384993.1 transcript variant22 XM_017024973.2 transcript variant 22 XM_024450896.1 *Each of which ishereby incorporated by reference in its entirety.

In some embodiments, the ZNF274 inhibitor, MAX inhibitor, E2F6inhibitor, IKZF3 inhibitor, and/or STAT3 inhibitor is a nuclease-basedgene editing system capable of silencing expression of ZNF274, MAX,E2F6, IKZF3, and/or STAT3. As used herein, the term “nuclease-based geneediting system” refers to a system comprising a nuclease or a derivativethereof that can be recruited to a target sequence in the genome. Thesystem may comprise a Clustered Regularly Interspaced Short PalindromicRepeat-associated (“Cas”) protein (e.g., Cas9, Cas12a, and Cas12b), azinc finger nuclease (“ZFNs”), or a transcription activator-likeeffector nucleases (“TALEN”).

In some embodiments, the nuclease-based gene editing system is aCRISPR/Cas system targeted to silence ZNF274 expression, MAX expression,E2F6 expression, IKZF3 expression, and/or STAT3 expression. TheCRISPR/Cas system may comprise a Cas protein or a nucleic acid moleculeencoding the Cas protein and a guide RNA comprising a nucleotidesequence that is complementary to a portion of a target DNA sequence.

As described herein, Cas proteins form a ribonucleoprotein complex witha guide RNA, which guides the Cas protein to a target DNA sequence.Suitable Cas proteins include Cas nucleases (i.e., Cas proteins capableof introducing a double strand break at a target nucleic acid sequence),Cas nickases (i.e., Cas protein derivatives capable of introducing asingle strand break at a target nucleic acid sequence), and nucleasedead Cas (dCas) proteins (i.e., Cas protein derivatives that do not haveany nuclease activity).

In some embodiments, the Cas protein is a Cas9 protein. As used herein,the term “Cas9 protein” or “Cas9” includes any of the recombinant ornaturally-occurring forms of the CRISPR-associated protein 9 (Cas9) orvariants or homologs thereof. In some embodiments, the variants orhomologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g., a 50, 100, 150, or 200 continuous amino acid portion) compared toa naturally occurring Cas9 protein. In some embodiments, the Cas9protein is substantially identical to the protein identified by theUniProt reference number Q99ZW2, G3ECR1, J7RUA5, A0Q5Y3, or J3F2B0(which are hereby incorporated by reference in their entirety) or avariant or homolog having substantial identity thereto. In someembodiments, the Cas9 protein is selected from the group consisting of aCas9 nuclease, a Cas9 nickases, and a nuclease dead Cas 9 (“dCas9”).

In some embodiments, the Cas protein is a Cas12a protein. As usedherein, the term “Cas12a protein” or “Cas12a” includes any of therecombinant or naturally-occurring forms of the CRISPR-associatedprotein 12 (Cas12a) or variants or homologs thereof. In someembodiments, the variants or homologs have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g., a 50, 100, 150, or 200 continuousamino acid portion) compared to a naturally occurring Cas12a protein. Insome embodiments, the Cas12a protein is substantially identical to theprotein identified by the UniProt reference number A0Q7Q2, U2UMQ6,A0A7C6JPC1, A0A7C9H0Z9, or A0A7J0AY55 (which are hereby incorporated byreference in their entirety) or a variant or homolog having substantialidentity thereto. In some embodiments, the Cas 12a protein is selectedfrom the group consisting of a Cas12a nuclease, a Cas12a nickase, and anuclease dead Cas12a (“dCas12a”).

In some embodiments, the Cas protein is a Cas12b protein. As usedherein, the term “Cas12b protein” or “Cas12b” includes any of therecombinant or naturally-occurring forms of the CRISPR-associatedprotein 12 (Cas12b) or variants or homologs thereof. In someembodiments, the variants or homologs have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g., a 50, 100, 150, or 200 continuousamino acid portion) compared to a naturally occurring Cas12b protein. Insome embodiments, the Cas12b protein is substantially identical to theprotein identified by the UniProt reference number T0D7A2, A0A6I3SPI6,A0A6I7FUC4, A0A6N9TP17, A0A6M1UF64, A0A7Y8V748, A0A7X7KIS4, A0A7X8X2U5,or A0A7X8UMW7 (which are hereby incorporated by reference in theirentirety) or a variant or homolog having substantial identity thereto.In some embodiments, the Cas 12b protein is selected from the groupconsisting of a Cas12b nuclease, a Cas12b nickase, and a nuclease deadCas12b (“dCas12b”).

As used herein, the term “guide RNA” or “gRNA” refers to aribonucleotide sequence capable of binding a nucleoprotein, therebyforming ribonucleoprotein complex. In accordance with the methods andsystems of the present disclosure, the guide RNA comprises (i) aDNA-targeting sequence that is complementary to a target nucleic acidsequence of ZNF274, MAX, E2F6, IKZF3, or STAT3 sequence and (ii) abinding sequence for the Cas protein (e.g., Cas9 nuclease, Cas9 nickase,dCas9, Cas12a nuclease, Cas12a nickase, or dCas12a).

In some embodiments, the guide RNA is a single guide RNA molecule(single RNA nucleic acid), which may include a “single-guide RNA” or“sgRNA”. In other embodiments, the nucleic acid of the presentdisclosure includes two RNA molecules (e.g., joined together viahybridization at the binding sequence). Thus, the term guide RNA isinclusive, referring both to two-molecule nucleic acids and to singlemolecule nucleic acids (e.g., sgRNAs).

In some embodiments, the gRNA is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100or more nucleic acid residues in length. In some embodiments, the gRNAis from 10 to 30 nucleic acid residues in length. In some embodiments,the gRNA is 20 nucleic acid residues in length. In some embodiments, thelength of the gRNA is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleic acidresidues or sugar residues in length. In some embodiments, the gRNA isfrom 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75,30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100,30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100,95 to 100, or more residues in length. In some embodiments, the gRNA isfrom 10 to 15, 10 to 20, 10 to 30, 10 to 40, or 10 to 50 residues inlength.

In some embodiments, a CRISPR/Cas system is targeted to silence ZNF274gene expression, and the guide RNA comprises a nucleotide sequence thatis complementary to a portion of a ZNF274 gene sequence.

In some embodiments, a CRISPR/Cas system is targeted to silence MAX geneexpression, and the guide RNA comprises a nucleotide sequence that iscomplementary to a portion of a MAX gene sequence.

In some embodiments, a CRISPR/Cas system is targeted to silence E2F6gene expression, and the guide RNA comprises a nucleotide sequence thatis complementary to a portion of a E2F6 gene sequence.

In some embodiments, a CRISPR/Cas system is targeted to silence IKZF3gene expression, and the guide RNA comprises a nucleotide sequence thatis complementary to a portion of a IKZF3 gene sequence.

In some embodiments, a CRISPR/Cas system is targeted to silence STAT3DNA expression, and the guide RNA comprises a nucleotide sequence thatis complementary to a portion of a STAT3 gene sequence.

In some embodiments, the CRISPR/Cas-system targeted to silence ZNF274expression, MAX expression, E2F6 expression, IKZF3 expression, and/orSTAT3 expression is a CRISPRi system. As used herein, the term “CRISPRinterference” or “CRISPRi” refers to a system that allows forsequence-specific repression of gene expression. CRISPRi systemscomprise nuclease dead Cas (“dCas”) proteins (i.e., nuclease-inactivatedCas proteins) to block the transcription of a target gene, withoutcutting the target DNA sequence. Nuclease inactivated Cas proteins andmethods of generating nuclease-inactivated Cas proteins are well knownin the art (see, e.g., Qi et al., “Repurposing CRISPR as an RNA-GuidedPlatform for Sequence-Specific Control of Gene Expression,” Cell152(5):1173-1183 (2013), which is hereby incorporated by reference inits entirety).

The CRISPRi system suitable for use as described herein may comprise (i)a nuclease dead Cas (dCas) protein (i.e., a nuclease-inactivated Casprotein) or nucleic acid molecule encoding the dCas protein and (ii) aguide RNA comprising a nucleotide sequence that is complementary to aportion of ZNF274, MAX, E2F6, IKZF3, and STAT3.

In some embodiments, the nuclease dead Cas (dCas) protein is selectedfrom the group consisting of dCas9, dCas12a, and dCas12b.

In some embodiments, the nuclease dead Cas (dCas) protein is a fusionprotein comprising a Cas protein and one or more epigenetic modulatorsthat suppress or silence the expression of the target gene, i.e.,ZNF274, MAX, E2F6, IKZF3, and STAT3.

Suitable epigenetic modulators include, but are not limited to, DNAmethyltransferase enzymes (e.g., DNA methyltransferase 3 alpha(“DNMT3A”) and DNA methyltransferase 3 like (“DNMT3L”)), histonedemethylation enzymes (e.g., lysine- specific histone demethylase 1(“LSD1”)), histone methyltransferase enzymes (e.g., G9A and SuV39h1),transcription factor recruitment domains (e.g., Kruppel-associated boxdomain (“KRAB”), KRAB-Methyl-CpG binding protein 2 domain(“KRAB-MeCP2”), enhancer of Zeste 2 (“EZH2”)), zinc fingertranscriptional repressor domains (e.g., spalt like transcription factor1 (“SALL1”) and suppressor of defective silencing protein 3 (“SDS3”))(see, e.g., Brezgin et al., “Dead Cas Systems: Types, Principles, andApplications,” Int. J. Mol. Sci. 20:6041 (2019), which is herebyincorporated by reference in its entirety).

In some embodiments, the epigenetic modulator is selected from the groupconsisting of DNMT3A, DNMT2L, LSD1, KRAB, KRAB-MeCP2, EZH2, SALL1, SDS3,G9A, and Suv39h1 (see, e.g., Yeo et al., “An Enhanced CRISPR Repressorfor Targeted Mammalian Gene Regulation,” 15(8):611-616 (2018); Alerasoolet al., “An Efficient KRAB Domain for CRISPRi Applications in HumanCells,” Nature Methods 17:1093-1096 (2020); and Duke et al., “AnImproved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression,”Frontiers in Genome Editing 2:9 (2020), which are hereby incorporated byreference in their entirety).

In some embodiments, the ZNF274 inhibitor is a CRISPRi system targetedto silence ZNF274 DNA expression, and the guide RNA comprises anucleotide sequence that is complementary to a portion of a ZNF274 genesequence.

In some embodiments, the MAX inhibitor is a CRISPRi system targeted tosilence MAX DNA expression, and the guide RNA comprises a nucleotidesequence that is complementary to a portion of a MAX gene sequence.

In some embodiments, the E2F6 inhibitor is a CRISPRi system targeted tosilence E2F6 DNA expression, and the guide RNA comprises a nucleotidesequence that is complementary to a portion of a E2F6 gene sequence.

In some embodiments, the IKZF3 inhibitor is a CRISPRi system targeted tosilence IKZF3 DNA expression, and the guide RNA comprises a nucleotidesequence that is complementary to a portion of a IKZF3 gene sequence.

In some embodiments, the STAT3 inhibitor is a CRISPRi system targeted tosilence STAT3 DNA expression, and the guide RNA comprises a nucleotidesequence that is complementary to a portion of a STAT3 gene sequence.

In some embodiments, the agent that suppresses ZNF274, MAX, E2F6, IKZF3and/or STAT3 comprises one or more expression vectors that express oneor more transcription factor inhibitors selected from the groupconsisting of ZNF274 inhibitors, MAX inhibitors, E2F6 inhibitors, IKZF3inhibitors and IKZF3 inhibitors. Each expression vector comprises (1) anucleotide sequence encoding one or more nucleic acid inhibitors forZNF274, MAX, E2F6, IKZF3 and/or IKZF3, and (2) a regulatory sequenceoperably linked to the nucleotide sequence. In some embodiments, theregulatory sequence comprises a glial cell specific promoter selectedfrom the group consisting of PDGFRA promoter, ZNF488 promoter, GPR17promoter, OLIG2 promoter, CSPG4 promoter, and SOX10 promoter. In someembodiments, the regulatory sequence comprises an inducible promoter orpromoter system, such as tetracycline controlled inducible system,cumate-controlled inducible system and rapamycin controlled induciblesystems, which are described in more detail infra.

In some embodiments, the one or more expression vectors comprises aplasmid vector, a viral vector, or a bacterial vector. In someembodiments, one or more expression vectors comprises a viral vectorselected from the group consisting of adenoviruses, AAV, retroviruses,lentiviruses, vaccinia viruses, and herpes viruses. In some embodiments,the one or more expression vectors comprises a lentiviral vector. Insome embodiments, the one or more expression vectors comprises aretroviral vector. In other embodiments, the one or more expressionvectors comprises an AAV vector. Methods for generating and isolatingviral vectors suitable for use as expression vectors are described inmore details infra.

IV. Expression Vectors

Another aspect of the disclosure relates to an expression vectordescribed in this application. In some embodiments, the expressionvector expresses one or more transcription factors selected from thegroup consisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2,wherein the expression vector comprises (1) a nucleotide sequenceencoding one or more transcription factors selected from the groupconsisting of BCL11A, HDAC2, EZH2, MYC, HMGA2, NFIB and TEAD2, and (2) aregulatory sequence operably linked to the nucleotide sequence

In some embodiments, the expression vector comprises (1) a nucleotidesequence encoding one or more nucleic acid inhibitors for ZNF274, MAX,E2F6, IKZF3 and/or IKZF3, and (2) a regulatory sequence operably linkedto the nucleotide sequence.

In some embodiments, the regulatory sequence comprises a glial cellspecific promoter selected from the group consisting of PDGFRA promoter,ZNF488 promoter, GPR17 promoter, OLIG2 promoter, CSPG4 promoter, andSOX10 promoter.

In some embodiments, the regulatory sequence comprises an induciblepromoter and/or operator system sequence. Inducible promoters and/oroperator systems that may be used in performing the methods or includedin the systems of the present disclosure include those regulated byhormones and hormone analogs such as progesterone, ecdysone andglucocorticoids as well as promoters which are regulated bytetracycline, heat shock, heavy metal ions, interferon, and lactoseoperon activating compounds. An inducible promoter and/operator systemis capable of directly or indirectly activating transcription of thenucleic acid molecule that it is operatively coupled to in response to a“regulatory agent” (e.g., a chemical agent or biological molecule, suchas a metabolite, a small molecule) or a stimulus. In the absence of a“regulatory agent” or stimulus, the nucleotide sequence operably linkedto the inducible promoter and/or operator system will not be transcribedor will not be substantially expressed.

The term “not be transcribed” or “not substantially expressed” meansthat the level of transcription is at least 50-fold lower than the levelof transcription observed in the presence of an appropriate stimulus orregulatory agent; and preferably at least 100 -fold, 250-fold, or500-fold or lower than the level of transcription observed in thepresence of an appropriate stimulus or regulatory agent. For a review ofthese systems see Gingrich & Roder, “Inducible Gene Expression in theNervous System of Transgenic Mice,” Annu. Rev. Neurosci. 21:377-405(1998), which is hereby incorporated by reference in its entirety.

Suitable inducible promoter and/or operator systems for inclusion in theexpression vector of the present disclosure are well known in the artand include, without limitation, tetracycline-controlled operatorsystems, cumate-controlled operator systems, rapamycin induciblesystems, FKCsA inducible system, and ABA inducible system (see, e.g.,Kallunki et al., “How to Choose the Right Inducible Gene ExpressionSystem for Mammalian Studies?” Cells 8(8):796 (2019); U.S. Pat. Nos.8,728,759; and 7,745,592, which are hereby incorporated by reference intheir entirety).

In some embodiments, the tetracycline-controlled operator systemcomprises a repression-based configuration, in which a Tet operator(“TetO”) is inserted between a constitutive promoter and gene ofinterest and where the binding of the Tet repressor (“TetR”) to theoperator suppresses downstream transcription of a nucleic acid sequenceof interest (see, e.g., Kallunki et al., “How to Choose the RightInducible Gene Expression System for Mammalian Studies?” Cells 8(8):796(2019), which is hereby incorporated by reference in its entirety). Inaccordance with such embodiments, the addition of tetracycline (or thesynthetic tetracycline derivative doxycycline) results in the disruptionof the association between TetR and TetO, thereby triggeringTetO-dependent transcription of the nucleic acid sequence of interest.

In some embodiments, the tetracycline-controlled operator systemcomprises a Tet-off configuration, where tandem TetO sequences arepositioned upstream of a minimal promoter followed by a nucleic acidsequence of interest (see, e.g., Kallunki et al., “How to Choose theRight Inducible Gene Expression System for Mammalian Studies?” Cells8(8):796 (2019), which is hereby incorporated by reference in itsentirety). In accordance with such embodiments, a chimeric proteinconsisting of TetR and VP16 (“tTA”), a eukaryotic transactivator derivedfrom herpes simplex virus type 1, is converted into a transcriptionalactivator, and the expression plasmid is transfected together with theoperator plasmid. Thus, the presence of tetracycline (or the synthetictetracycline derivative doxycycline) switches off the expression of thesystem or its components, while removing tetracycline switches it on.

In some embodiments, the tetracycline-controlled operator systemcomprises a Tet-on configuration, where the system or its components istranscribed when tetracycline is present (see, e.g., Kallunki et al.,“How to Choose the Right Inducible Gene Expression System for MammalianStudies?” Cells 8(8):796 (2019), which is hereby incorporated byreference in its entirety). In accordance with such embodiments, tandemTetO sequences are positioned upstream of a minimal promoter followed bya nucleic acid sequence of interest. In the presence of tetracycline (orthe 5 synthetic tetracycline derivative doxycycline), a mutant rTa(“rtTa”) binds to TetO sequences, thereby activating the minimalpromoter.

In some embodiments, the inducible promoter and/or operator system is acumate-controlled operator system. Similar to thetetracycline-controlled operator system, the cumate-controlled operatorsystem, the cumate operator (“CuO”) and its repressor (“CymR”) may beengineered into a repressor configuration, an activator configuration,and a reverse activator configuration (see, e.g., Kallunki et al., “Howto Choose the Right Inducible Gene Expression System for MammalianStudies?” Cells 8(8):796 (2019), which is hereby incorporated byreference in its entirety).

In some embodiments, the cumate-controlled operator system comprises arepression-based configuration, in which the cumate operator (“CuO”) isinserted between a constitutive promoter and gene of interest and wherethe binding of the cumate repressor (“CymR”) to the operator suppressesdownstream transcription of the system (or system components) asdescribed herein. In accordance with such embodiments, the addition ofcumate releases CymR thereby triggering CuO-dependent expression of thesystem or system components.

In some embodiments, the cumate-controlled operator system comprises anactivator configuration, where chimeric molecular (“cTA”) is formed viathe fusion of CymR and VP16. In this configuration, a minimal promoteris placed downstream of the multimerized operator binding sites (e.g.,6×CuO). Transcription of a nucleic acid sequence of interest iscontrolled by the minimal promoter, which is activated in the absence ofcumate.

In some embodiments, the cumate-controlled operator system comprises areverse activator configuration, where a nucleic acid sequence istranscribed when cumate is present. In accordance with such embodiments,tandem CuO sequences are positioned upstream of a minimal promoterfollowed by a nucleic acid sequence of interest. In the presence ofcumate, a cTA mutant (“rcTA”) binds to CuO sequences, thereby activatingthe minimal promoter.

In some embodiments, the inducible promoter and/or operator system is arapamycin inducible system. In accordance with such embodiments, thepromoter is a rapamycin-inducible promoter (e.g., a minimal IL-2promoter). In this system, a DNA 25 binding domain (ZFHD1) and atranscription factor activation domain (NF-KB p65) are expressedseparately as fusion proteins with the rapamycin-binding domains ofFKBP12 and FRAP (mTOR), respectively (see, e.g., Koh et al., “Use of aStringent Dimerizer-Regulated Gene Expression System for Controlled BMP2Delivery,” Mol. Ther. 14(5):P684-691 (2006), which is herebyincorporated by reference in its entirety). On addition of rapamycin (orthe rapamycin analog FK506), the fusion proteins are reversiblycross-linked to drive transcription of a nucleic acid sequence ofinterest. Mutation of the rapamycin-binding region of themTOR-activation domain fusion protein results in systems responsive torapamycin-like compounds (rapalogs) that, unlike rapamycin, do not bindto endogenous mTOR protein and, therefore, have little immunosuppressiveor antiproliferative activity.

In some embodiments, the regulatory sequence comprises a humanelongation factor la promoter (“EF1A”), 5 cytomegalovirus (“CMV”)promoter, human ubiquitin C promoter (“UBC”), chicken beta-actinpromoter, hybrid chicken beta-actin promoter (CBh) andphosphoglycerokinase (“PGK”) promoter. In accordance with suchembodiments, the promoter may be modified to comprise one, two, three,or more Tet operator (TetO) sites or one, two, three, four, five, six,or more CuO sites

Additional suitable promoters for inclusion in the expression vectors ofthe present application include, but are not limited to, H1 promoter andU6 promoter. In some embodiments, when the inducible promoter and/oroperator system is operatively linked to the nucleotide sequenceencoding one or more guide RNAs in a Cas system, the promoter is an H1promoter or a U6 promoter. The H1 promoter may be a modified H1 promotercomprising one, two, three, or more Tet operator (TetO) sites. The U6promoter may be a modified U6 promoter comprising one, two, or three Tetoperator (TetO) sites or one, two, three, four, five, six, or more CuOsites (see, e.g., Sun et al., “Development of Drug-20 InducibleCRISPR-Cas9 Systems for Large-Scale Functional Screening,” BMC Genomics20:225 (2019), which is hereby incorporated by reference in itsentirety).

In some embodiments, the regulatory sequence may further comprise atranscriptional enhancer binding site, a RNA polymerase initiation site,a ribosome binding site, and/or other sites that facilitate theexpression of the nucleotide sequence operably linked to the regulatorysequence in the expression vector.

In some embodiments, the expression vector encodes a system for inducingrejuvenation of a population of adult glial progenitor cells or treatingmyelin deficiency in a subject.

In some embodiments, the system for inducing rejuvenation of apopulation of adult glial progenitor cells or treating myelin deficiencyin a subject is a nuclease dead Cas (dCas) system. This system comprisesa first nucleic acid molecule encoding a fusion protein comprising anuclease dead Cas (dCas) protein fused to an epigenetic modulator; asecond nucleic acid molecule encoding one or more guide RNAs, where theone or more guide RNAs each comprise an RNA sequence that hybridizes toa portion of a DNA sequence of ZNF274, MAX, E2F6, IKZF3, and/or STAT3;and an inducible promoter and/or operator system that is operativelylinked to the first nucleic acid molecule, the second nucleic acidmolecule, or both. In some embodiments, the inactivated Cas protein(dCas) is selected from the group consisting of dCas9, dCas12a, anddCas12b (see, e.g., Brezgin et al., “Dead Cas Systems: Types,Principles, and Applications,” Int. J. Mol. Sci. 20:6041 (2019), whichis hereby incorporated by reference in its entirety).

Suitable epigenetic modulators are described in detail supra. In someembodiments, the epigenetic modulator is selected from the groupconsisting of DNMT3A, DNMT2L, LSD1, KRAB, KRAB-MeCP2, EZH2, SALL1, SDS3,G9A, Suv39h1, Cs, and WRPW (see, e.g., Yeo et al., “An Enhanced CRISPRRepressor for Targeted Mammalian Gene Regulation,” 15(8):611-616 (2018);Alerasool et al., “An Efficient KRAB Domain for CRISPRi Applications inHuman Cells,” Nature Methods 17:1093-1096 (2020); and Duke et al., “AnImproved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression,”Frontiers in Genome Editing 2:9 (2020), which are hereby incorporated byreference in their entirety). In some embodiments, when methylation of agene or gene promoter is effective to suppress its transcription, theepigenetic modulator is a methyltransferase.

In some embodiments, the epigenetic modulator is selected from the groupconsisting of Tet methylcytosine dioxygenase 1(“TET1”), SunTag-TET1,MS2/MCP- TET1, p300Core, four tandem copies of herpes simplex viralprotein 16 (“VP64”), VP160, NF-KB p65 activation domain (“p65”),Epstein-Barr Virus-derived R transactivator (“Rta”), SunTag-VP64,VP64-p65-Rta (“VPR”), SunTag-p65-HSF1, TV, synergistic activationmediator (“SAM”), Three-Component Repurposed Technology for EnhancedExpression (“TREE”), Casilio, Scaffold, and CMV (see, e.g., Brezgin etal., “Dead Cas Systems: Types, Principles, and Applications,” Int. J.Mol. Sci. 20(23):6041 (2019), which is hereby incorporated by referencein its entirety). In some embodiments, when demethylation of a gene orgene protein is effective to suppress its transcription, the epigeneticmodulator is a demethylase (e.g., TET1).

In some embodiments, the system for inducing rejuvenation in apopulation of adult glial progenitor cells or treating myelin deficiencyin a subject comprises a dCas fusion protein, where the dCas is fused toa methyltransferase. In any embodiment, the system for inducingrejuvenation in a population of adult glial progenitor cell or treatingmyelin deficiency in a subject comprises a dCas fusion protein, wherethe dCas is fused to a demethylase.

Exemplary dCas fusion proteins and dCas fusion protein systems for useaccording to the methods of the present disclosure are identified inTable 9 below.

TABLE 9 Exemplary Cas Fusion Proteins dCas9-TET1 Liu et al., “EditingDNA Methylation in the Mammalian Genome,” Cell 167: 233-247 (2016)dCas9-SunTag- Morita et al., “Targeted DNA Demethylation in vivo TET1using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions,” Nat.Biotechnol. 34: 1060-1065 (2016) dCas9-MS2/ Xu et al., “A CRISPR-BasedApproach for Targeted MCP-TET1 DNA Demethylation,” Cell Discov. 2: 16009(2016) dCas9-P300 Hilton et al., “Epigenome Editing by a CoreCRISPR/Cas9-Based Acetyltransferase Activates Genes from Promoter andEnhancers,” Nat. Biotechnol. 33(5): 510-517 (2015) dCas9-VP64 Gilbert etal., “CRISPR-Mediated Modular RNA-Guided Regulation of Transcription inEukaryotes,” Cell 154: 442-451 (2013) dCas9-VP160 Cheng et al.,“Multiplexed Activators of Endogenous Genes by CRISPR-on, an RNA-GuidedTranscriptional Activator System,” Cell Res. 23: 1163 (2013) dCas9-p65Gilbert et al., “CRISPR-Mediated Modular RNA-Guided Regulation ofTranscription in Eukaryotes,” Cell 154: 442-451 (2013) dCas9-VPR Chavezet al., “Highly Efficient Cas9-Mediated Transcriptional Programming,”Nat. Methods. 12: 326 (2015) dCas9 SAM Konermann et al., “Genome-ScaleTranscriptional system Activation by an Engineered CRISPR-Cas9 Complex,”Nature 517(7536): 583-588 (2015) dCas9 TREE Kunii et al.,“Three-Component Repurposed system Technology for Enhanced Expression:Highly Accumulable Transcriptional Activators via Branched Tag Arrays,”CRISPR J. 1(5): 337-347 (2018) dCas9-TV (i.e., Li et al., “A potentCas9-Derived Gene Activator for dCas9-6TAL- Plant and Mammalian Cells,”Nat. Plants. 3: 930-936 VP128) (2017) dCas12a-VPR Liu et al.,“Engineering Cell Signaling Using Tunable CRISPR-Cpf1-BasedTranscription Factors,” Nat. Commun. 8: 2095 (2017) dCas12a-p65 Tak etal., “Inducible and Multiplex Gene Regulation Using CRISPR-Cpf1-BasedTranscription Factors,” Nat. Methods 14: 1163-1166(2017) *Each of whichis hereby incorporated by reference in its entirety.

In some embodiments, the dCas protein is a dCas9 or dCas 12 protein.

In some embodiments, the first and second nucleic acid molecules of thisdCas system are contained in a single expression vector. In someembodiments, the first and second nucleic acid molecules of this systemare contained in separate expression vectors.

In some embodiments, the system for inducing rejuvenation of apopulation of adult glial progenitor cells or treating myelin deficiencyin a subject is a Cas system. This system comprises a first nucleic acidmolecule encoding a Cas protein; a second nucleic acid molecule encodingone or more guide RNAs, where said one or more guide RNAs each comprisean RNA sequence that hybridizes to a portion of a DNA sequence ofZNF274, MAX, E2F6, IKZF3, and/or STAT3; and an inducible promoter and/oroperator system that is operatively linked to the first nucleic acidmolecule, the second nucleic acid molecule, or both.

In some embodiments, this Cas nuclease system comprises a Cas9 proteinor a Cas12 protein (e.g., Cas12a or Cas12b). Suitable Cas proteins andderivatives thereof for inclusion in the systems according to thepresent disclosure are well known in the art and are described in moredetail supra.

In some embodiments, the first and second nucleic acid molecules of thisCas nuclease system are contained in a single expression vector. In someembodiments, the first and second nucleic acid molecules are containedin separate expression vectors.

In some embodiments, the system for inducing rejuvenation of apopulation of adult glial progenitor cells or treating myelin deficiencyin a subject is a gene-editing nuclease system. This system comprises: afirst nucleic acid molecule encoding a first sequence specific geneediting nuclease comprising a first DNA binding motif, where the firstDNA binding motif binds to a first DNA sequence of ZNF274, MAX, E2F6,IKZF3 and/or STAT3, respectively; a second nucleic acid moleculeencoding a second sequence specific gene editing nuclease comprising asecond DNA binding motif, where said second DNA binding motif binds to asecond DNA sequence of ZNF274, MAX, E2F6, IKZF3 and/or STAT3,respectively; and an inducible promoter and/or operator system sequencethat is operatively coupled to the first nucleic acid molecule, thesecond nucleic acid molecule, or both the first and second nucleic acidmolecules.

Suitable sequence specific gene editing nucleases for inclusion thesystems according to the present disclosure are well known in the artand include, without limitation, zinc finger nucleases (“ZFNs”) andtranscription activator-like effector nucleases (“TALENs”).

In one embodiment, the sequence specific gene editing nucleases of thesystem described herein is a ZFN. A ZFN is an artificial endonucleasethat comprises at least 1 zinc finger motif (e.g., at least 2, 3, 4, or5 zinc finger motifs) fused to a nuclease domain (e.g., the cleavagedomain of the FokI restriction enzyme). Heterodimerization of twoindividual ZFNs at a target nucleic acid sequence can result in cleavageof the target sequence. For example, two individual ZFNs may bindopposite strands of a target DNA sequence to induce a double-strandbreak in the target nucleic acid sequence. Methods of designing suitableZFNs for inclusion in the systems of the presently claimed disclosureare well known in the art (see, e.g., Urnov et al., “Genome Editing withEngineered Zinc Finger Nucleases,” Nat. Rev. Genet. 11(9):636-646(2010); Gaj et al., “Targeted Gene Knockout by Direct Delivery ofZinc-Finger Nuclease Proteins,” Nat. Methods 9(8):805-807 (2012); U.S.Pat. Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978;6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719;7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; and 6,479,626,which are hereby incorporated by reference in their entirety). In someembodiments, the first and second gene editing nucleases are FokInucleases. In accordance with such embodiments, the first and second DNAbinding motifs are zinc finger motifs.

In some embodiments, the sequence specific gene editing nucleases of thesystem described herein is a TALEN. A TALEN is an engineeredtranscription activator-like effector nuclease that comprise aDNA-binding domain and a nuclease domain (e.g., a cleavage domain of theFokI restriction enzyme). The DNA-binding domain comprises a series of33-35 amino acid repeat domains that each recognize a single base pair.Heterodimerization of two individual TALENs at a target nucleic acidsequence can result in cleavage of the target sequence. For example, twoindividual TALENs may bind opposite strands of a target DNA sequence toinduce a double-strand break in the target nucleic acid sequence.Methods of designing suitable ZFNs for inclusion in the systems of thepresently claimed disclosure are well known in the art (see, e.g.,Scharenberg et al., “Genome Engineering with TAL-Effector Nucleases andAlternative Modular Nuclease Technologies,” Curr. Gene Ther.13(4):291-303 (2013); Gaj et al., “Targeted Gene Knockout by DirectDelivery of Zinc-Finger Nuclease Proteins,” Nat. Methods 9(8):805-807(2012); Beurdeley et al., “Compact Designer TALENs for Efficient GenomeEngineering,” Nat. Commun. 4:1762 (2013); U.S. Pat. Nos. 8,440,431;8,440,432; 8,450,471; 8,586,363; and 8,697,853, which are herebyincorporated by reference in their entirety). In some embodiments, thefirst and second gene editing nucleases are FokI nucleases. Inaccordance with such embodiments, the first and second DNA bindingmotifs are TALE motifs.

In some embodiments, the first and second nucleic acid molecules of thesequence specific gene editing nuclease systems described herein arecontained in a single expression vector. In some embodiments, the firstand second nucleic acid molecules are contained in separate expressionvectors.

In all systems described herein for rejuvenating glial progenitor cellsor treating myelin deficiency in a subject, the first and/or secondnucleic acid molecules of the system are operatively coupled to aninducible promoter and/or operator system as described in more detailsabove.

In some embodiments, the expression vector of the present application isa plasmid vector, a viral vector, or a bacterial vector.

In some embodiments, the expression vector of the present application isa lentiviral vector (see, e.g., U.S. Pat. No. 748,529 to Fang et al.;Ura et al., “Developments in Viral Vector-Based Vaccines,” Vaccines 2:624-641 (2014); and Hu et al., “Immunization Delivered by LentiviralVectors for Cancer and Infection Diseases,” Immunol. Rev. 239: 45-61(2011), 15 which are hereby incorporated by reference in theirentirety).

In some embodiments, the expression vector of the present application isa retroviral vector (see e.g., U.S. Pat.No. 748,529 to Fang et al., andUra et al., “Developments in Viral Vector-Based Vaccines,” Vaccines 2:624-641 (2014), which are hereby incorporated by reference in theirentirety), a vaccinia virus, a replication deficient adenovirus vector,and a gutless adenovirus vector (see e.g., U.S. Pat. No. 5,872,005,which is incorporated herein by reference in its entirety).

In other embodiments, the expression vector of the present applicationis an adeno-associated virus (AAV) vector (see, e.g., Krause et al.,“Delivery of Antigens by Viral Vectors for Vaccination,” Ther. Deliv.2(1):51-70 (2011); Ura et al., “Developments in Viral Vector-BasedVaccines,” Vaccines 2: 624-641 (2014); Buning et al, “RecentDevelopments in Adeno-associated Virus Vector Technology,” J. Gene Med.10:717-733 (2008), each of which is incorporated herein by reference inits entirety).

Methods for generating and isolating viral expression vectors suitablefor use as vectors are known in the art (see, e.g., Bulcha et al.,“Viral Vector Platforms within the Gene Therapy Landscape,” Nature 6:53(2021); Bouard et al., “Viral Vectors: From Virology to TransgeneExpression,” Br. J. Pharmacol. 157(2):153-165 (2009); Grieger &Samulski, “Adeno-associated Virus as a Gene Therapy Vector: VectorDevelopment, Production and Clinical Applications,” Adv. Biochem.Engin/Biotechnol. 99: 119-145 (2005); Buning et al, “Recent Developmentsin Adeno-associated Virus Vector Technology,” J. Gene Med. 10:717-733(2008), each of which is incorporated herein by reference in itsentirety).

V. Genetically Modified Glial Progenitor Cell

Aspects of the present disclosure also relate to a glial progenitor cellgenetically modified with an expression vector or nucleic acid moleculeof the present application. In some embodiments, the expression vectoror nucleic acid molecule is integrated into the genome of thegenetically modified glial progenitor cell. In some embodiments, theexpression vector or nucleic acid molecule is present in anepichromosomal form in the genetically modified glial progenitor cell.

In some embodiments, the glial progenitor cell is genetically modifiedwith a nuclease dead Cas (dCas) system, a Cas nuclease system, or agene-editing nuclease system according to the present application.

In some embodiments, the genetically modified glial progenitor cell is amammalian glial progenitor cell. In any embodiment, the geneticallymodified glial progenitor cell is a human glial progenitor cell.

Glial progenitor cells suitable for genetic modification as describedhere can be derived from multipotent (e.g., neural stem cells) orpluripotent cells (e.g., embryonic stem cells and induced pluripotentstem cells) using methods known in the art or described herein. In yetanother embodiment, glial progenitor cells can be extracted fromembryonic tissue, fetal tissue, or adult brain tissue containing a mixedpopulation of cells directly by using the promoter specific separationtechnique, as described in U.S. Patent Application Publication Nos.20040029269 and 20030223972 to Goldman, which are hereby incorporated byreference in their entirety. In accordance with this embodiment, theglial progenitor cells are isolated from ventricular or subventricularzones of the brain or from the subcortical white matter.

In some embodiments, the genetically modified glial progenitor cells aregenetically modified bi-potential glial progenitor cells. In someembodiment, the genetically modified glial progenitor cells aregenetically modified oligodendrocyte-biased glial progenitor cells. Insome embodiments, the genetically modified glial progenitor cells aregenetically modified astrocyte-biased glial progenitor cells.

In some embodiment, it may be preferable to enrich a cell preparationcomprising glial progenitor cells prior to or after genetic modificationto increase the concentration and/or purity of the glial progenitorcells modified to contain the expression vector or systems describedherein. Accordingly, in one embodiment, A2B5 monoclonal antibody (mAb)that recognizes and binds to gangliosides present on glial progenitorcells early in the developmental or differentiation process is utilizedto separate glial progenitor cells from a mixed population of cells(Nunes et al., “Identification and Isolation of Multipotential NeuralProgenitor Cells From the Subcortical White Matter of the Adult HumanBrain.,” Nat Med. 9(4):439-47 (2003), which is hereby incorporated byreference in its entirety). Using the A2B5 mAb, glial progenitor cellscan be separated, enriched, or purified from a mixed population of celltypes. In another embodiment, selection of CD140α/PDGFRα positive cellsis employed to produce a purified or enriched preparation ofbi-potential glial progenitor cells. In another embodiment, selection ofCD9 positive cells is employed to produce a purified or enrichedpreparation of oligodendrocyte-biased glial progenitor cells. In yetanother embodiment, both CD140α/PDGFRα and CD9 positive cell selectionis employed to produce a purified or enriched preparation ofoligodendrocyte-biased glial progenitor cells. In another embodiment,selection of CD44 positive cells is employed to produce a purified orenriched preparation of astrocyte-biased glial progenitor cells (Liu etal., “CD44 Expression Identifies Astrocyte-Restricted Precursor Cells,”Dev. Biol. 276(1):31-46 (2004), which is hereby incorporated byreference in its entirety.) In another embodiment, both CD140α/PDGFRαand CD44 positive cell selection is employed to produce a purified orenriched preparation of oligodendrocyte-biased glial progenitor cells.In another embodiment, CD140α/PDGFRα, CD9, and CD44 positive cellselection is employed to produce a purified or enriched preparation ofoligodendrocyte-biased glialprogenitor cells.

A further aspect of the present disclosure is directed to a preparationof glial progenitor cells expressing the genetic construct according tothe present disclosure.

The examples below are intended to exemplify the practice of embodimentsof the disclosure but are by no means intended to limit the scopethereof.

EXAMPLES Materials and Methods

Cell Lines

The human iPSC line C27 was used to generate hGPCs in which predictedtranscripts of interest were validated. The C27 line is male. Cells weredifferentiated into GPCs as detailed in Human iPSC-derived production ofGPCs (Chambers et al., “Highly Efficient Neural Conversion of Human ESand iPS Cells by Dual Inhibition of SMAD Signaling,” Nat Biotechnol27:275-280 (2009), which is hereby incorporated by reference in itsentirety).

Adult and Fetal Brain Processing for Cell Isolation

Human brain samples were obtained under approved Institutional ReviewBoard protocols from consenting patients at Strong Memorial Hospital atthe University of Rochester. Brain tissue was obtained from normal GW18-24 cortical and/or VZ/SVZ dissections or adult white matter/cortexepileptic resections (18F,19M, and 27F years old for mRNA, 8M, 20F, 43M,and 54F years old for miRNA). Fetal GPC acquisition, dissociation andimmunomagnetic sorting of A2B5+/PSA-NCAM− cells were as described(Windrem et al., “Fetal and Adult Human Oligodendrocyte Progenitor CellIsolates Myelinate the Congenitally Dysmyelinated Brain,” Nat. Med.10:93-97 (2004), which is hereby incorporated by reference in itsentirety). GPCs were isolated from dissociated tissue using a dualimmunomagnetic sorting strategy: depleting mouse anti-PSA-NCAM+(Millipore, DSHB) cells, using microbead tagged rat anti-mouse IgM(Miltenyi Biotech), then selecting A2B5+ (clone 105; ATCC, Manassas,Va.) cells from the PSA-NCAM− pool, as described (Windrem et al., “Fetaland Adult Human Oligodendrocyte Progenitor Cell Isolates Myelinate theCongenitally Dysmyelinated Brain,” Nat. Med. 10:93-97 (2004) and Windremet al., “Neonatal Chimerization with Human Glial Progenitor Cells canboth Remyelinate and Rescue the Otherwise Lethally HypomyelinatedShiverer Mouse,” Cell Stem Cell 2:553-565 (2008), which are herebyincorporated by reference in their entirety). After sorting, cells weremaintained for 1-14 days in DMEM-F12/N1 with 10 ng/ml bFGF and 20 ng/mlPDGF-AA. Alternatively, CD140a/PDGF□R-defined GPCs were isolated andsorted using MACS as previously described (Sim et al., “CD140aIdentifies a Population of Highly Myelinogenic, Migration-Competent andEfficiently Engrafting Human Oligodendrocyte Progenitor Cells,” Nat.Biotechnol. 29:934-941 (2011b), which is hereby incorporated byreference in its entirety), yielding an enriched population of CD140+glial progenitor cells.

Bulk RNA-Sequencing

RNA was purified from isolates via Qiagen RNeasy kits and bulk RNAsequencing libraries were constructed. Samples were sequenced deeply onan Illumina HiSeq 2500 at the University of Rochester Genomics ResearchCenter. Raw FASTQ files were trimmed and adapters removed using fastp(Chen et al., “fastp: An Ultra-fast All-in-one FASTQ Preprocessor,”Bioinformatics 34:i884-i890 (2018), which is hereby incorporated byreference in its entirety) and aligned to GRCh38 using Ensembl 95 geneannotations via STAR in 2-pass mode across all samples (Dobin et al.,“STAR: Ultrafast Universal RNA-seq Aligner,” Bioinformatics 29:15-21(2013), which is hereby incorporated by reference in its entirety) andquantified with RSEM version (Li and Dewey, “RSEM: Accurate TranscriptQuantification From RNA-Seq Data With or Without a Reference Genome,”BMC Bioinformatics 12:323 (2011), which is hereby incorporated byreference in its entirety). Subsequent analysis was carried out in R (RCore Team R: A Language and Environment for Statistical Computing.(Vienna, Austria: R Foundation for Statistical Computing) (2017), whichis hereby incorporated by reference in its entirety) where RSEM genelevel results were imported via tximport (Soneson et al., “DifferentialAnalyses for RNA-seq: Transcript-Level Estimates Improve Gene-LevelInferences,” F1000Research 4:1521 (2015), which is hereby incorporatedby reference in its entirety). DE analysis was carried out in DESeq2(Love et al., “Moderated Estimation of Fold Change and Dispersion forRNA-seq Data With DESeq2,” Genome Biology 15:550 (2014), which is herebyincorporated by reference in its entirety) where paired analyses (FetalA2B5+ vs CD140a+, fetal CD140a+ vs CD140a−) had paired information addedto their models. For adult vs fetal DE analysis, age was concatenatedwith sort marker (CD140a− samples were not included) to define the groupvariable where sequencing batch was also added to the model to accountfor technical variability. Genes with an adjusted p-value<0.01 and anabsolute log2-fold change>1 were deemed significant. These data werethen further filtered by meaningful abundance, defined as a median TPM(calculated via RSEM) of 1 in at least 1 group (20,663 genes met thiscriterion prior to DE).

scRNA-Seq Analysis

The fetal brain sample as processed as above for bulk rna-seq up untilsingle cells were sorted via FACS for either CD140a+ or PSA-NCAM−/A2B5+surface expression. Single cells were then captured on a 10× genomicschromium controller utilizing V2 chemistry and libraries generatedaccording to manufacturer's instructions.

Samples were sequenced on an Illumina HISeq 2500 system. Demultiplexedsamples were then aligned and quantified using Cell Ranger to an indexgenerated from GRCh38 and Ensembl 95 gene annotations using only proteincoding, lncRNA, or miRNA biotypes. Analysis of scRNA-Seq samples wascarried out via Seurat (Butler et al., “Integrating Single-cellTranscriptomic Data Across Different Conditions, Technologies, andSpecies,” Nat Biotechnol 36:411-420 (2018), which is hereby incorporatedby reference in its entirety) within R. Both samples were merged andlow-quality cells filtered out as defined by having mitochondrial geneexpression greater than 15% or having fewer than 500 unique genes.Samples were then normalized utilizing SCTransform taking care toregress out contributions due to total number of UMIs, percentmitochondrial gene content or the difference in S phase and G2M phasescores of each cell. PCA was then calculated, UMAP was run using thefirst 30 dimensions with n.neighbors=60 and repulsion.strength=0.8.FindNeighbors was then run followed by FindClusters with resolution setto 0.35. Based on expression profiles of each cluster, some similarclusters were merged into broader cell type clusters. Staticdifferential expression of clusters was computed using the MAST test(Finak et al., “MAST: A Flexible Statistical Framework for AssessingTranscriptional Changes and Characterizing Heterogeneity in Single-cellRNA Sequencing Data,” Genome Biology 16:278 (2015), which is herebyincorporated by reference in its entirety) where an adjusted p-value of<0.01 and an absolute log2 fold change of >0.5 was deemed significant.Prediction of active transcription factor regulons was carried out withthe SCENIC package in R (Aibar et al., “SCENIC: Single-cell RegulatoryNetwork Inference and Clustering,” Nat. Methods 14:1083-1086 (2017),which is hereby incorporated by reference in its entirety) using thehg38 databases located at https://resources.aertslab.org/cistarget/.Genes were included in co-expression analyses if they were expressed inat least 1% of cells.

Ingenuity Pathway Analysis and Network Construction

Differentially expressed genes were fed into Ingenuity Pathway Analysis(Qiagen) to determine significant canonical, functional, and upstreamsignaling terms. For construction of the IPA network, terms werefiltered for adjusted p-values below 0.001. Non-relevant IPA terms wereremoved along with highly redundant functional terms assessed viajaccard similarity indices using the iGraph package (Csardi, G.N., Tamas“The Igraph Software Package for Complex Network Research,” InterJournalComplex Systems 1695 (2006), which is hereby incorporated by referencein its entirety). Modularity was established within Gephi (Bastian etal., “Gephi: An Open Source Software for Exploring and ManipulatingNetworks,” (2009), which is hereby incorporated by reference in itsentirety) and the final network was visualized using Cytoscape (ShannonP., “Cytoscape: A Software Environment for Integrated Models ofBiomolecular Interaction Networks,” Genome Res 13:2498-2504 (2003),which is hereby incorporated by reference in its entirety). Genes andterms of interest were retained for visualization purposes. Modules werebroken out from one another and organized using the yFiles organiclayout.

Inference of Transcription Factor Activity

Adult and fetal enriched gene lists were fed separately into RcisTarget(Aibar et al., “SCENIC: Single-cell Regulatory Network Inference andClustering,” Nat. Methods 14:1083-1086 (2017), which is herebyincorporated by reference in its entirety) to identifyoverrepresentation of motifs in windows around the genes' promoters(500bp up/100bp down and 10kb up and 10kb down). Transcription factorsthat were associated with significantly enriched motifs (NES>3) werethen filtered by their significant differential expression in the inputgene list. Within each window and gene list, only appropriate TF-geneinteractions (Repressors downregulating genes and activatorsupregulating genes) were kept. Scanning windows were then merged toproduce TF-gene edge lists of predicted fetal/adultrepressors/activators. TFs of interest were narrowed to those primarilyreported as solely activators or repressors in the literature.

miRNA Microarray Analysis

A2B5+adult (n=3) and CD140a+ fetal (n=4) cell suspensions were isolatedvia MACS as noted above and their miRNA isolated using miRNeasy kitsaccording to manufacturer instructions (QIAGEN). Purified miRNA was thenprepared and profiled on Affymetrix GeneChip miRNA 3.0 Arrays asinstructed by their standard protocol. Raw CEL files were then read intoR via the oligo (Carvalho and Irizarry, “A Framework for OligonucleotideMicroarray Preprocessing,” Bioinformatics 26:2363-2367 (2010), which ishereby incorporated by reference in its entirety) package and sampleswere normalized via robust multi-array averaging (RMA). Probes were thenfiltered for only human miRNAs according to Affymetrix's annotation, anddifferential expression was carried out in limma (Ritchie et al.,“LimmaPowers Differential Expression Analyses for RNA-Sequencing andMicroarray Studies,” Nucleic Acids Res 43:e47 (2015), which is herebyincorporated by reference in its entirety) where significance wasestablished at an adjusted p-value <0.01. Finally, differentiallyexpressed miRNAs were surveyed across five independent miRNA predictiondatabases using miRNAtap (Pajak M., “miRNAtap: miRNAtap: microRNATargets-Aggregated Predictions,” R Package Version 1.22.0 (2020), whichis hereby incorporated by reference in its entirety) with min_src set to2 and method set to “geom”. Transcription factor regulation of miRNAswas carried out via querying the TrasmiR V2.0 database (Tong et al.,“TransmiR v2.0: An Updated Transcription Factor-microRNA RegulationDatabase,” Nucleic Acids Res 47:D253-D258 (2019), which is herebyincorporated by reference in its entirety).

Exploratory Analysis and Visualization

PCA of bulk RNA-Seq or microarray samples was computed via prcomp withdefault settings on variance stabilized values of DESeq2 objects. PCAswere plotted via autoplot in the ggfortify package. Volcano plots weregenerated using EnhancedVolcano. Graphs were further edited or generatedanew using ggplot2 and aligned using patchwork.

Human iPSC-derived production of GPCs

Human induced pluripotent stem cells (C27 (Chambers et al., “HighlyEfficient Neural Conversion of Human ES and iPS Cells by Dual Inhibitionof SMAD Signaling,” Nat Biotechnol 27:275-280 (2009), which is herebyincorporated by reference in its entirety)) were differentiated intoGPCs using our previously described protocol (Osipovitch et al., “HumanESC-Derived Chimeric Mouse Models of Huntington's Disease RevealCell-Intrinsic Defects in Glial Progenitor Cell Differentiation,” CellStem Cell 24:107-122 e107 (2019); Wang et al., “Human iPSC-DerivedOligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Modelof Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013); andWindrem et al., “Human iPSC Glial Mouse Chimeras Reveal GlialContributions to Schizophrenia,” Cell Stem Cell 21:195-208.e196 (2017),which are hereby incorporated by reference in their entirety). Briefly,cells were first differentiated to neuroepithelial cells, then topre-GPCs, and finally to GPCs. GPCs were maintained in glial mediasupplemented with T3, NT3, IGF1, and PDGF-AA.

Lentiviral Overexpression

For overexpression of E2F6, ZNF274, IKZF3, or MAX, we first identifiedthe most abundant protein coding transcript of each of these genes fromthe adult hGPC dataset. cDNAs for each transcript were cloned downstreamof the tetracycline response element promoter in thepTANK-TRE-EGFP-CAG-rtTA3G-WPRE vector. Viral particles pseudotyped withvesicular stomatitis virus G glycoprotein were produced by transienttransfection of HEK293FT cells and concentrated by ultracentrifugation,and titrated by QPCR (qPCR Lentivirus Titer Kit, ABM-Applied BiologicalMaterials Inc). iPSC (C27) derived GPC cultures (160-180 days in vitro)were infected at 1.0 MOI in glial media for 24 hours. Cells were washedwith HBSS and maintained in glial media supplemented with 1 μg/mldoxycycline (Millipore-Sigma St. Louis, Mo.) for the remainder of theexperiment. Transduced hGPCs were isolated via FACS onDAPI-/EGFP+expression 3, 7, and 10 days following the initial additionof doxycycline. Doxycycline control cells were sorted on DAPI− alone.

Quantitative PCR

RNA from overexpression experiments was extracted using RNeasy microkits (Qiagen, Germany). First-strand cDNA was synthesized using TaqManReverse Transcription Reagents (Applied Biosystems, USA). qPCR reactionswere run in triplicate by loading 1 ng of RNA mixed with FastStartUniversal SybrGreen Mastermix (Roche Diagnostics, Germany) per reactionand analyzed on a real-time PCR instrument (CFX Connect Real-Time Systemthermocycler; Bio-Rad). Results were normalized to the expression of 18Sfrom each sample.

Quantification and Statistical Analysis

For qPCR experiments, significant differences in delta CTs for each genewere analyzed in linear models constructed by the interaction ofoverexpression condition and timepoint with the addition of a cell batchcovariate. Post hoc pairwise comparisons were tested via least-squaresmeans tests against the Dox control within timepoints using the lsmeanspackage (Lenth, R. V., “Least-Squares Means: The R Package lsmeans,”Journal of Statistical Software, Foundation for Open Access Statistics69(i01) (2016), which is hereby incorporated by reference in itsentirety). P-values were adjusted for multiple comparisons using thefalse discovery rate method whereby p-values <0.05 were deemedsignificant.

Example 1: CD140a Selection Enriches for Human Fetal Glial Progenitorsmore Efficiently than Does A2B5

To identify the transcriptional concomitants to GPC aging, bulk andsingle cell RNA-Seq was first used to characterize hGPCs derived fromsecond trimester fetal human tissue, whether isolated by targeting theCD140a epitope of PDGFRα (Sim et al., “CD140a Identifies a Population ofHighly Myelinogenic, Migration-Competent and Efficiently EngraftingHuman Oligodendrocyte Progenitor Cells,” Nature Biotechnology 29:934-941(2011a), which is hereby incorporated by reference in its entirety), orthe glial gangliosides recognized by monoclonal antibody A2B5 (Dietrichet al., “Characterization of A2B5+ Glial Precursor Cells FromCryopreserved Human Fetal Brain Progenitor Cells,” Glia 40:65-77 (2002);Sim et al., “CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nature Biotechnology 29:934-941 (2011a); and Windremet al., “Fetal and Adult Human Oligodendrocyte Progenitor Cell IsolatesMyelinate the Congenitally Dysmyelinated Brain,” Nat. Med. 10:93-97(2004), which are hereby incorporated by reference in their entirety).To that end, two sample-matched experiments were carried out whereby theventricular/subventricular zones (VZ/SVZ) of 18-22-week gestational age(g.a.) fetal brains were dissociated and sorted via fluorescenceactivated cell sorting (FACS), for either CD140a+ and A2B5+/PSA-NCAM−(A2B5+) GPCs isolated from the same fetal brain (n=3), or for CD140a+GPCs as well as the CD140a-depleted remainder (n=5; FIG. 1 , Panel A).Bulk RNA-Seq libraries were then generated and deeply sequenced for bothexperiments. Principal component analysis (PCA) showed segregation ofthe CD140a+ and A2B5+ cells, and further segregation of both from theCD140a-depleted samples (FIG. 1 , Panel B). Differential expression inboth paired cohorts (p<0.01, absolute log2 fold change>1) identified 723genes as differentially-expressed between CD140a+ and A2B5+ GPCs (435 inCD140a, 288 in A2B5, Table S1). In contrast, 2,629 genes distinguishedCD140a+ GPCs from CD140a− cells (FIG. 1 , Panel C). Differential geneexpression directionality was highly consistent when comparing CD140+ toeither A2B5+ or CD140− cells, with all but 4 genes being concordant.

Pathway enrichment analysis using Ingenuity Pathway Analysis (IPA) ofboth of these gene sets identified similar pathways as relatively activein CD140+ GPCs; these pathways included cell movement, oligodendroglialdifferentiation, lipid synthesis, and downstream PDGF, SOX10, and TCF7L2signaling (FIG. 1 , Panel D). As expected, stronger activation Z-scoreswere typically observed when comparing CD140a+ GPCs to CD140a− cellsrather than to A2B5+ GPCs. Interestingly, CD140a+ cells alsodifferentially expressed a number of pathways related to the immunesystem, likely due to small amounts of microglial contamination as aresult of re-expression of PDGF□R epitopes on the microglial surface.A2B5+ samples additionally displayed upregulated ST8SIA1, the enzymeresponsible for A2B5 synthesis (Sim et al., “Fate Determination of AdultHuman Glial Progenitor Cells,” Neuron Glia Biol 5:45-55 (2009), which ishereby incorporated by reference in its entirety), as well as pro-neuralpathways.

Among the genes differentially upregulated in CD140a+ isolates werePDGFRA itself, and a number of early oligodendroglial genes includingOLIG1, OLIG2, NKX2-2, SOX10, and GPR17 (FIGS. 1 , Panel E-1, Panel F).Furthermore, the CD140a+ fraction also exhibited increased expression oflater myelinogenesis-associated genes, including MBP, GAL3ST1, and UGT8.Beyond enrichment of the oligodendroglial lineage, many genes typicallyassociated with microglia were also enriched in the CD140a isolates,including CD68, C2, C3, C4, and TREM2. In contrast, A2B5+ isolatesexhibited enrichment of astrocytic (AQ4, CLU) and early neuronal(NEUROD1, NEUROD2, GABRG1, GABRA4, EOMES, HTR2A) genes, suggesting theexpression of A2B5 by immature astrocytes and neurons as well as by GPCsand oligodendroglial lineage cells. Overall then, oligodendroglialenrichment was significantly greater in CD140a+ GPCs than A2B5-definedGPCs, when each was compared to depleted fractions, suggesting theCD140a isolates as being the more enriched in hGPCs, and thus CD140a asthe more appropriate phenotype for head-to-head comparison with adulthGPCs.

Example 2: Single Cell RNA-Sequencing Reveals Cellular Heterogeneitywithin Human Fetal GPC Isolates

To further delineate the composition of fetal hGPC isolates at singlecell resolution, both CD140a+ and A2B5+ hGPCs were isolated from 20-weekg.a. fetal VZ/SVZ via FACS, and then the transcriptomes of each wereassayed by single cell RNA-Seq (FIG. 1 , Panel A, 10X Genomics V2). Itwas sought to capture>1,000 cells of each; following filtration oflow-quality cells (unique genes<500, mitochondrial gene percentage>15%),1,053 PSA-NCAM−/A2B5+ and 957 CD140a+ high quality cells remained(median 6,845 unique molecular identifiers and 2,336 unique genes percell). Dimensional reduction via uniform manifold approximation andprojection (UMAP), followed by shared nearest neighbor modularity-basedclustering of all cells using Seurat (Butler et al., “IntegratingSingle-cell Transcriptomic Data Across Different Conditions,Technologies, and Species,” Nat Biotechnol 36:411-420 (2018), which ishereby incorporated by reference in its entirety), revealed 11 clusterswith 8 primary cell types, as defined by their differential enrichmentof marker genes. These primary cell types included: GPCs, pre-GPCs,neural progenitor cells (NPCs), immature neurons, neurons, microglia,and a cluster consisting of endothelial cells and pericytes. We foundthat the CD140a+ FACS isolates were more enriched for GPC and pre-GPCpopulations than were the fetal A2B5+/PSA-NCAM− cells (FIGS. 2 , PanelA-2, Panel D). Furthermore, whereas the CD140a− sorted cells werelargely limited to GPCs and pre-GPCs, with only scattered microglialcontamination, the A2B5+/PSA-NCAM− isolates also included astrocytes andneuronal lineage cells, the latter despite the upfront depletion ofneuronal PSA-NCAM. These data supported the more selective andphenotypically-restricted nature of CD140a rather than A2B5-based GPCisolation.

On that basis, the gene expression profiles of the predominant cellpopulations in the CD140a+ fetal isolates, GPCs and pre-GPCs, was nextexplored. Differential expression between these two pools yielded 269(143 upregulated, 126 down-regulated; p<0.01, 1og2 fold change>0.5; FIG.2 , Panel E). During the pre-GPC to GPC transition, earlyoligodendroglial lineage genes were rapidly upregulated (OLIG2, SOX10,NKX2-2, PLLP, APOD), whereas those expressed in pre-GPCs effectivelydisappeared (VIM, HOPX, TAGLN2, TNC). Interestingly, genes involved inthe human leukocyte antigen system, including HLA-A, HLA-B, HLA-C andB2M, were all downregulated as the cells transitioned to GPC stage (FIG.2 , Panel F). IPA analysis indicated that pre-GPCs were relativelyenriched for terms related to migration, proliferation, and thosepresaging astrocytic identity (BMP4, AGT, and VEGF signaling), whereasGPCs displayed enrichment for terms associated with acquisition of anoligodendroglial identity (PDGF-AA, FGFR2, CCND1), in addition toactivation of the MYC and MYCN pathways (FIG. 2 , Panel G). Using singlecell co-expression data together with promoter motif enrichment usingthe SCENIC package (Aibar et al., “SCENIC: Single-cell RegulatoryNetwork Inference and Clustering,” Nat Methods 14:1083-1086 (2017),which is hereby incorporated by reference in its entirety), 262transcription factors that were predicted to be relatively activated inGPCs vs pre- GPCs were next identified (Wilcoxon rank sum test, p<0.01).These included SATB1, as well as the early GPC specification factorsOLIG2, SOX10, and NKX2-2 (FIG. 2 , Panel H).

Example 3: Human Adult and Fetal GPCs are Transcriptionally Distinct

The study next asked how adult hGPCs might differ in their transcriptionfrom fetal hGPCs. To this end, A2B5+hGPCs were isolated fromsurgically-resected adult human temporal neocortex (19-21 years old,n=3) and their bulk RNA expression assessed, as paired together withfour additional fetal CD140a+ samples. It had been previously noted thatA2B5 selection is sufficient to isolate GPCs from adult human brain, andis more sensitive than CD140a in that regard, given thematuration-associated down-regulation of PDGFRA expression in adulthGPCs (Sim et al., “Complementary Patterns of Gene Expression by HumanOligodendrocyte Progenitors and their Environment Predict Determinantsof Progenitor Maintenance and Differentiation,” Ann Neurol 59:763-779(2006) and Windrem et al., “Fetal and Adult Human OligodendrocyteProgenitor Cell Isolates Myelinate the Congenitally DysmyelinatedBrain,” Nat. Med. 10:93-97 (2004), which are hereby incorporated byreference in their entirety).

Confirming that prior observation, it was found that PDGFRA in A2B5+adult GPCs was expressed with a median TPM of 0.55, compared to a medianTPM of 47.56 for fetal A2B5+ cells. By pairing the sequencing andanalysis with fetal CD140a-selected cells, regression of sequencingbatch effects was enabled while simultaneously increasing power (FIG. 3, Panel A). Depletion of PSA-NCAM+ cells was not necessary for adulthGPC samples, as the expression of PSA-NCAM ceases in the adult cortexand white matter (Seki et al., “Distribution and Possible Roles of theHighly Polysialylated Neural Cell Adhesion Molecule (NCAM-H) in theDeveloping and Adult Central Nervous System,” Neurosci. Res. 1:265-290(1993), which is hereby incorporated by reference in its entirety). As aresult, PCA of human adult and fetal GPCs illustrated tight clusteringof adult GPCs, sharply segregated from both sorted fetal hGPC pools(FIG. 3 , Panel B). Differential expression of adult GPCs compared toeither A2B5+ or CD140a+ fetal GPC populations yielded 3,142 and 5,282significant genes, respectively (p<0.01; absolute log2 fold-change>1)(FIG. 3 , Panel C). To increase the accuracy of defining differentialexpression, downstream analyses were carried out on the intersecting2,720 genes (FIG. 3 , Panel D, 1,060 up-regulated and 1,660down-regulated in adult GPCs, compared to fetal hGPCs). Remarkably,within these two differentially-expressed gene sets, 100% of genes weredirectionally concordant.

To better understand the differences between adult and fetal GPCs, agene ontology network of non-redundant significant IPA terms and theircontributing differentially-expressed genes was constructed (FIGS. 3 ,Panel D-3, Panel E). Spin glass community detection of this network(Reichardt et al., “Statistical Mechanics of Community Detection,” Phys.Rev. E Stat. Nonlin. Soft Matter Phys. 74:016110 (2006), which is herebyincorporated in its entirety) uncovered three modules (Modules M1-M3) ofhighly connected functional terms (FIG. 3E) and genes (FIG. 3F, TableS3). M1 included terms and genes linked to glial development,proliferation, and movement. Notably, a number of genes associated withGPC ontogeny were downregulated in adult GPCs; these included CSPG4/NG2,PCDH15, CHRDL1, LMNB1, PTPRZ1, and ST8SIA1 (Huang et al., “Origins andProliferative States of Human Oligodendrocyte Precursor Cells,” Cell182:594-608 e511 (2020); McClain et al., “Pleiotrophin Suppression ofReceptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-renewalCompetence of Fetal Human Oligodendrocyte Progenitor Cells,” J Neurosci32:15066-15075 (2012); Nishiyama et al., “Co-Localization of NG2Proteoglycan and PDGF α-Receptor on 02A Progenitor Cells in theDeveloping Rat Brain,” Journal of Neuroscience Research 43:299-314(1996); Sim et al., “Fate Determination of Adult Human Glial ProgenitorCells,” Neuron Glia Biol 5:45-55 (2009); and Yattah et al., “DynamicLamin B 1-Gene Association During Oligodendrocyte ProgenitorDifferentiation,” Neurochem Res 45:606-619 (2020), which are herebyincorporated by reference in their entirety). In contrast, numerousgenes whose appearance precedes and continues through oligodendrocytedifferentiation and myelination were upregulated in adult GPCs,including MAG, MOG, MYRF, PLP1, CD9, CLDN11, CNP, ERBB4, GJB1, PMP22,and SEMA4D.

Module 2 harbored numerous terms associated with cellular aging and themodulation of proliferation and senescence. Cell cycle progression andmitosis were predicted to be activated in fetal GPCs due to strongenrichment of proliferative factors including MKI67, TOP2A, CENPF,CENPH, CHEK1, EZH2 and numerous cyclins, including CDK1 and CDK4.Furthermore, proliferation-inducing pathways were also inferred to beactivated; these included MYC, CCND1, and YAP1 signaling, of which bothYAP1 and MYC transcripts were similarly upregulated (Bretones et al.,“Myc and Cell Cycle Control,” Biochim. Biophys Acta 1849:506- 516(2015); Bunt et al., “Regulation of Cell Cycle Genes and Induction ofSenescence by Overexpression of OTX2 in Medulloblastoma Cell Lines,”Mol. Cancer Res. 8:1344-1357 (2010); and Xie et al., “YAP/TEAD-MediatedTranscription Controls Cellular Senescence,” Cancer Res 73:3615-3624(2013), which are hereby incorporated by reference in their entirety).In that regard, transient overexpression of MYC in aged rodent GPCs hasrecently been shown to restore their capacity to both proliferate anddifferentiate (Neumann et al., “Myc Determines the Functional Age Stateof Oligodendrocyte Progenitor Cells,” Nature Aging 1:826-837 (2021),which is hereby incorporated by reference in its entirety). Conversely,adult GPCs exhibited an upregulation of senescence-associatedtranscripts, including E2F6, MAP3K7, DMTF1/DMP1, OGT, AHR, RUNX1, andRUNX2 (Ferrand et al., “Screening of a Kinase Library Reveals NovelPro-senescence Kinases and Their Common NF-κB-dependent TranscriptionalProgram,” Aging (Albany N.Y.) 7:986-1003 (2015); Inoue et al.,“Disruption of the ARF Transcriptional Activator DMP1 Facilitates CellImmortalization, Ras Transformation, and Tumorigenesis,” Genes Dev14:1797-1809 (2000); Lee and Zhang, “O-Linked N-AcetylglucosamineTransferase (OGT) Interacts With the Histone Chaperone HIRA Complex andRegulates Nucleosome Assembly and Cellular Senescence,” Proceedings ofthe National Academy of Sciences 113:E3213-E3220 (2016); Wotton et al.,“RUNX1 Transformation of Primary Embryonic Fibroblasts is Revealed inthe Absence of p53,” Oncogene 23:5476-5486 (2004); and Kilbey et al.,“Runx2 Disruption Promotes Immortalization and Confers Resistance toOncogene-induced Senescence in Primary Murine Fibroblasts,” Cancer Res67:11263- 11271 (2007), which are hereby incorporated by reference intheir entirety). At the same time, adult hGPCs exhibited adown-regulation of fetal transcripts that included LMNB1, PATZ1, BCL11A,HDAC2, FN1, EZH2, and YAP1 and its cofactor TEAD1 (Cho et al., “POZ/BTBand AT-hook-containing Zinc Finger Protein 1 (PATZ1) InhibitsEndothelial Cell Senescence Through a p53 Dependent Pathway,” Cell DeathDiffer 19:703-712 (2012); Fan et al., “EZH2-dependent Suppression of aCellular Senescence Phenotype in Melanoma Cells by Inhibition ofp21/CDKN1A Expression,” Mol. Cancer Res. 9:418-429 (2011); Freund etal., “Lamin B1 Loss is a Senescence-associated Biomarker,” Mol. Biol.Cell 23:2066-2075 (2012); Luc et al., “Bcl11a Deficiency Leads toHematopoietic Stem Cell Defects with an Aging-like Phenotype,” Cell Rep.16:3181-3194 (2016); Lukjanenko et al., “Loss of Fibronectin From theAged Stem Cell Niche Affects the Regenerative Capacity of SkeletalMuscle in Mice,” Nat Med 22:897-905 (2016); Sundar et al., “GeneticAblation of Histone Deacetylase 2 Leads to Lung Cellular Senescence andLymphoid Follicle Formation in COPD/Emphysema,” FASEB Journal: OfficialPublication of the Federation of American Societies for ExperimentalBiology 32:4955-4971 (2018); and Xie et al., “YAP/TEAD-MediatedTranscription Controls Cellular Senescence,” Cancer Res 73:3615-3624(2013), which are hereby incorporated by reference in their entirety).As a result, functional terms predicted to be active in adult hGPCsincluded senescence, the rapid onset of aging observed inHutchinson-Gilford progeria, and cyclin-dependent kinase inhibitorypathways downstream of CDKN1A/p21 and CDKN2A/p16. Furthermore, AHR andits signaling pathway, which has been implicated in driving senescencevia the inhibition of MYC (Yang et al., “The Aryl Hydrocarbon ReceptorConstitutively Represses C-Myc Transcription in Human Mammary TumorCells,” Oncogene 24:7869-7881 (2005), which is hereby incorporated byreference in its entirety), was similarly upregulated in adult GPCs.

Module 3 consisted primarily of developmental and disease linkedsignaling pathways that have also been associated with aging. Thisincluded the predicted activation of ASCL1 and BDNF signaling in fetalhGPCs and MAPT/Tau, APP, and REST signaling in adult GPCs (Ahlin et al.,“High Expression of Cyclin D1 is Associated to High Proliferation Rateand Increased Risk of Mortality in Women With ER-positive But Not inER-negative Breast Cancers,” Breast Cancer Res. Treat 164:667-678(2017); Erickson et al., “Brain-derived Neurotrophic Factor isAssociated With Age- related Decline in Hippocampal Volume,” J.Neurosci. 30:5368-5375 (2010); and Harris et al., “Coordinated Changesin Cellular Behavior Ensure the Lifelong Maintenance of the HippocampalStem Cell Population,” Cell Stem Cell (2021), which are herebyincorporated by reference in their entirety). Overall, thetranscriptional and functional profiling of adult GPCs revealed areduction in transcripts associated with proliferative capacity, and ashift toward senescence and more mature phenotype.

Example 4: Inference of Transcription Factor Activity Implicates AdultGPC Transcriptional Repressors

Given the significant transcriptional disparity between adult and fetalGPCs, the study next asked whether it could infer which transcriptionfactors direct their identities. To accomplish this, two promoterwindows (500 bp up/100 bp down, 10 kb up/10 kb down) of adult or fetalenriched GPC gene sets were first scanned to infer significantlyenriched TF motifs (Aibar et al., “SCENIC: Single-cell RegulatoryNetwork Inference and Clustering,” Nat. Methods 14:1083-1086 (2017),which is hereby incorporated by reference in its entirety). Thisidentified 48 TFs that were also differentially-expressed in the scannedintersecting dataset. Among these, TFs whose primary means of DNAinteraction were exclusively either repressive or stimulatory, werefirst investigated, while also considering the enrichment of their knowncofactors. This analysis yielded 12 potential upstream regulators toexplore (FIGS. 4 , Panel A-4, Panel C): 4 adult repressors, E2F6,ZNF274, MAX, and IKZF3; 1 adult activator, STAT3; 3 fetal repressors,BCL11A HDAC2, and EZH2; and 4 fetal activators, MYC, HMGA2, NFIB, andTEAD2. Interestingly, of these predicted TFs, 3 groups shared a highconcordance of motif similarity within their targeted promoters: 1)E2F6, ZNF274, MAX, and MYC; 2) STAT3 and BCL11A; and 3) EZH2 and HDAC2,suggesting that they may cooperate or compete for DNA binding at sharedloci (FIG. 4 , Panel A).

Next, four potential signaling pathways were constructed based oncurated transcriptional interactions, to predict those genes targeted bythe set of identified TFs (FIG. 4 , Panel D-4, Panel G). Amongactivators enriched in fetal GPCs (FIG. 4 , Panel D), MYC, aproliferative factor (Dang, C.V., “c-Myc Target Genes Involved in CellGrowth, Apoptosis, and Metabolism,” Molecular and Cellular Biology 19:1(1999), which is hereby incorporated by reference in its entirety),NFIB, a key determinant of gliogenesis (Deneen et al., “TheTranscription Factor NFIA Controls the Onset of Gliogenesis in theDeveloping Spinal Cord,” Neuron 52:953-968 (2006), which is herebyincorporated by reference in its entirety), TEAD2, a YAP/TAZ effector,and HMGA2, another proliferative factor, were each predicted to activatecohorts of progenitor stage genes, including both mitogenesis-associatedtranscripts and those demonstrated to inhibit the onset of senescence(Dang, C. V., “c-Myc Target Genes Involved in Cell Growth, Apoptosis,and Metabolism,” Molecular and Cellular Biology 19:1 (1999); Deneen etal., “The Transcription Factor NFIA Controls the Onset of Gliogenesis inthe Developing Spinal Cord,” Neuron 52:953-968 (2006); Diepenbruck etal., “Tead2 Expression Levels Control the Subcellular Distribution ofYap and Taz, Zyxin Expression and Epithelial-mesenchymal Transition,”Journal of Cell Science 127:1523-1536 (2014); and Yu et al., “HMGA2Regulates the in Vitro Aging and Proliferation of Human Umbilical CordBlood-Derived Stromal Cells Through the mTOR/p70S6K Signaling Pathway,”Stem Cell Res 10:156-165 (2013), which are hereby incorporated byreference in their entirety). Direct positive regulation was alsopredicted between these four fetal activators, with NFIB being driven byHMGA2 and TEAD2, MYC being driven by TEAD2 and NFIB, HMGA2 being drivenby MYC and TEAD2, and TEAD2 being reciprocally driven by MYC (FIG. 4 ,Panel D). In contrast to these fetal activators, fetal stage repressors,including the C2H2 type zinc finger BCL11A, the polycomb repressivecomplex subunit EZH2, and histone deacetylase HDAC2, were each predictedto repress more mature oligodendrocytic gene expression at this stage(FIG. 4 , Panel E) (Laherty et al., “Histone Deacetylases AssociatedWith the mSin3 Corepressor Mediate Mad Transcriptional Repression,” Cell89:349-356 (1997); Laible et al., “Mammalian Homologues of thePolycomb-group Gene Enhancer of Zeste Mediate Gene Silencing inDrosophila Heterochromatin and at S. cerevisiae Telomeres,” EMBO J16:3219-3232 (1997); and Nakamura et al., “Evi9 Encodes a Novel ZincFinger Protein that Physically Interacts with BCL6, a known Human B-CellProto-Oncogene Product,” Mol Cell Biol 20:3178-3186 (2000), which arehereby incorporated by reference in their entirety). Furthermore, allthree of these TFs were predicted to inhibit targets implicated insenescence. As such, these factors appear to directly orchestratedownstream transcriptional events leading to maintenance of the cyclingprogenitor state.

Next, these predicted adult GPC signaling networks were assessed for apotential mechanism responsible for their age-related gene expressionchanges. STAT3 was predicted to shift GPC identity towards glialmaturation via the upregulation of a large cohort of earlydifferentiation- and myelination-associated oligodendrocytic genes (FIG.4 , Panel F). In addition, STAT3 was also inferred to activate a set ofsenescence- associated genes including BIN1, RUNX1, RUNX2, DMTF1, CD47,MAP3K7, CTNNA1, and OGT. At the same time, repression in adult GPCs waspredicted to be effected through the Ikaros family zinc fingerIKZF3/Aiolos, the KRAB (kruppel associated box) zinc finger ZNF274, theMYC-associated factor MAX, and cell cycle regulator E2F6 (FIG. 4 , PanelG) (Blackwood and Eisenman, “Max: A Helix-loop-helix Zipper Protein ThatForms a Sequence-specific DNA-binding Complex With Myc,” Science251:1211-1217 (1991); Frietze et al., “ZNF274 Recruits the HistoneMethyltransferase SETDB1 to the 3′ Ends of ZNF Genes,” PLoS One 5:e15082(2010); Ma et al., “Ikaros and Aiolos Inhibit Pre-B-cell Proliferationby Directly Suppressing c-Myc Expression,” Mol Cell Biol 30:4149-4158(2010b); and Ogawa et al., “A Complex with Chromatin Modifiers thatOccupies E2F- and Myc-Responsive Genes in G0 Cells,” Science296:1132-1136 (2002), which are hereby incorporated by reference intheir entirety). Targeting by this set of transcription factorspredicted repression of those gene sets contributing to the fetal GPCsignature, and this was indeed observed in the down-regulation of theearly progenitor genes PDGFRA and CSPG4, as well as of the cellcyclicity genes CDK1, CDK4, and MKI67. Repression of YAP1, LMNB1, andTEAD1, whose expression slows or prevents the onset of senescence, wasalso predicted. Interestingly, this set of four adult repressorspredicted the down-regulated expression of each of the fetal enrichedactivators NFIB, MYC, TEAD2, and HMGA2, in addition to the fetalenriched repressors BCL11A, EZH2, and HDAC2.

Example 5: Expression of Adult-Enriched Repressors InducesAge-Associated Transcriptional Changes in GPCs

It was next asked whether the four adult-enriched transcriptionalrepressors identified in FIG. 4 , Panel G, E2F6, IKZF3, MAX, and ZNF274,were individually sufficient to induce aspects of the age-associatedchanges in gene expression by otherwise young GPCs. To accomplish this,doxycycline (Dox) inducible overexpression lentiviruses were designedfor each transcription factor (FIG. 5 , Panel A). Briefly, whichprotein-coding isoform was most abundant in adult GPCs for eachrepressor was first identified, so as to best mimic endogenousage-associated upregulation; these candidates were E2F6-202, IKZF3-217,MAX-201, and ZNF274-201. These cDNAs were cloned downstream of atetracycline response element promoter, and upstream of a T2Aself-cleaving EGFP reporter (FIG. 5 , Panel A). Human inducedpluripotent stem cell (iPSC)- derived hGPC cultures, prepared from theC27 line as previously described (Wang et al., “Human iPSC-DerivedOligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Modelof Congenital Hypomyelination,” Cell Stem Cell 12:252-264 (2013), whichis hereby incorporated by reference in its entirety), were then infectedfor 24 hours, and then treated with Dox to induce transgeneoverexpression. C27 iPSC-derived GPCs were chosen as their transcriptomeresembles that of fetal, and they are similarly capable of engraftingand myelinating dysmyelinated mice upon transplantation (Wang et al.,“Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate andRescue a Mouse Model of Congenital Hypomyelination,” Cell Stem Cell12:252-264 (2013) and Windrem et al., “Human iPSC Glial Mouse ChimerasReveal Glial Contributions to Schizophrenia,” Cell Stem Cell21:195-208.e196 (2017), which are hereby incorporated by reference intheir entirety). Over-expressing cells were selected via FACS for EGFPexpression, at 3, 7, and 10 days following Dox addition (FIG. 5 , PanelB, n=3-5). Uninfected cultures given Dox were used as controls.

RNA was extracted and aging-associated genes of interest were analyzedby qPCR. Significant induction of each adult-enriched repressor wasobserved at each timepoint following Dox supplementation (FIG. 5 , PanelC). MKI67 and CDK1, genes whose upregulation are associated with activecell division, were significantly repressed at two or more timepoints ineach over-expression paradigm (FIG. 5 , Panel D). This was consistentwith their diminished expression in adult GPCs (FIG. 3 , Panel F), andsuggested their direct repression by E2F6, MAX, and ZNF274 (MKI67), orby all four (CDK1). The GPC stage marker PDGFRA, the cognate receptorfor PDGF-AA, was also significantly repressed at two timepoints in theIKZF3-transduced GPCs, as well as in the E2F6- transduced GPCs at day 3,consistent with its repression in normal adult GPCs. Interestingly, thesenescence-associated cyclin-dependent kinase inhibitor CDKN1A/p21 wasupregulated in response to each of the tested repressors at alltimepoints, while CDKN2A/p16 was similarly upregulated in at alltimepoints in ZNF274-transduced hGPCs, as well as in theE2F6-over-expressing GPCs at day 7 (FIG. 5 , Panel D). In addition, MBPand ILIA, both of which are strongly upregulated in adult hGPCs relativeto fetal, both exhibited sharp trends towards upregulated expression inresponse to repressor transduction, although timepoint-associatedvariability prevented their increments from achieving statisticalsignificance. Together, these data supported our prediction that forced,premature expression of the adult-enriched GPC repressors, E2F6, IKZF3,MAX, and ZNF274, are individually sufficient to induce multiple featuresof the aged GPC transcriptome in young, iPSC-derived GPCs.

Example 6: The miRNA Expression Pattern of Fetal hGPCs Predicts TheirSuppression of Senescence

To identify potential post-transcriptional regulators of geneexpression, we assessed differences in miRNA expression between adultand fetal GPCs (n=4) utilizing Affymetrix GeneChip miRNA 3.0 arrays. PCAdisplayed segregation of both GPC populations as defined by their miRNAexpression profiles (FIG. 6 , Panel A). Differential expression betweenboth ages (adjusted p-value<0.01) yielded 56 genes (23 enriched in adultGPCs, 33 enriched in fetal GPCs, FIG. 6 , Panel B-C). Notably amongthese differentially expressed miRNAs were the adult oligodendrocyteregulators miR-219a-3p and miR-338-5p (Dugas, J. C., Cuellar, T. L.,Scholze, A., Ason, B., Ibrahim, A., Emery, B., Zamanian, J. L., Foo, L.C., McManus, M. T., and Barres, B.A. (2010). Dicerl and miR-219 Arerequired for normal oligodendrocyte differentiation and myelination.Neuron 65, 597-611) in addition to fetal progenitor stage miRNAsmiR-9-3p, miR-9-5p (Lau, P., Verrier, J. D., Nielsen, J. A., Johnson, K.R., Notterpek, L., and Hudson, L. D. (2008). Identification ofdynamically regulated microRNA and mRNA networks in developingoligodendrocytes. J Neurosci 28, 11720-11730), and miR-17-5p (Budde, H.,Schmitt, S., Fitzner, D., Opitz, L., Salinas-Riester, G., and Simons, M.(2010). Control of oligodendroglial cell number by the miR-17-92cluster. Development 137, 2127).

The study next utilized this cohort of miRNAs to predict genes whoseexpression might be expected to be repressed via miRNA upregulation,separately analyzing both the adult and fetal GPC pools. To accomplishthis, miRNAtap were used to query five miRNA gene target databases:DIANA (Maragkakis, M., Vergoulis, T., Alexiou, P., Reczko, M.,Plomaritou, K., Gousis, M., Kourtis, K., Koziris, N., Dalamagas, T., andHatzigeorgiou, A. G. (2011). DIANA-microT Web server upgrade supportsFly and Worm miRNA target prediction and bibliographic miRNA to diseaseassociation. Nucleic Acids Res 39, W145-148), Miranda (Enright, A. J.,John, B., Gaul, U., Tuschl, T., Sander, C., and Marks, D. S. (2003).MicroRNA targets in Drosophila. Genome biology 5, R1), PicTar (Lall, S.,Grun, D., Krek, A., Chen, K., Wang, Y. L., Dewey, C. N., Sood, P.,Colombo, T., Bray, N., Macmenamin, P., et al. (2006). A genome-wide mapof conserved microRNA targets in C. elegans. Current biology : CB 16,460-471), TargetScan (Friedman, R. C., Farh, K. K., Burge, C. B., andBartel, D. P. (2009). Most mammalian mRNAs are conserved targets ofmicroRNAs. Genome Res 19, 92-105), and miRDB (Wong, N., and Wang, X.(2015). miRDB: an online resource for microRNA target prediction andfunctional annotations. Nucleic Acids Res 43, D146-152).

To maximize precision, genes were only considered a target if theyappeared in at least two databases. Among fetal-enriched miRNAs, thisapproach predicted an average of 36.3 (SD=24.5) repressed genes permiRNA. In contrast, among adult hGPC-enriched miRNAs, an average of 46.4(SD=37.8) genes were predicted as targets per miRNA (FIG. 6 , Panel C).Altogether, this identified the potential repression of 48.8% of adultGPC-enriched genes via fetal miRNAs, and repression of 39.9% of fetalGPC-enriched genes by adult miRNAs.

To assess the functional importance of these miRNA-dependentpost-transcriptional regulatory mechanisms, the study curated fetal andadult networks according to miRNA targeting of functionally-relevant,differentially expressed genes (FIG. 6 , Panel D-E). The proposedupstream adult transcriptional regulators STAT3, E2F6, and MAX werepredicted to be inhibited via 7 miRNAs in fetal GPCs (FIG. 24 , PanelD); these included the already-validated repression of STAT3 in othercell types by miR-126b-5p, miR-106a-5p, miR-7-5p, miR-130a-3p, andmiR-130b-3p (Du, W., Pan, Z., Chen, X., Wang, L., Zhang, Y., Li, S.,Liang, H., Xu, C., Zhang, Y., Wu, Y., et al. (2014a). By Targeting Stat3microRNA-17-5p Promotes Cardiomyocyte Apoptosis in Response to IschemiaFollowed by Reperfusion. Cellular Physiology and Biochemistry 34,955-965). In parallel, a number of early and mature oligodendrocyticgenes were concurrently targeted for inhibition, all consistent withmaintenance of the progenitor state; these included MBP, UGT8, CD9,PLP1, MYRF, and PMP22 (Goldman, S. A., and Kuypers, N.J. (2015). How tomake an oligodendrocyte. Development 142, 3983-3995). Importantly, acohort of genes linked to either the induction of senescence orinhibition of proliferation, or both, were also predicted to be activelyrepressed in fetal GPCs. These included RUNX1, RUNX2, BIN1, DMTF1/DMP1,CTNNA1, SERPINE1, CDKN1C, PAK1, IFI16, EFEMP1, MAP3K7, AHR, OGT, CBX7,and CYLD (Eckers, A., Jakob, S., Heiss, C., Haarmann-Stemmann, T., Goy,C., Brinkmann, V., Cortese-Krott, M.M., Sansone, R., Esser, C.,Ale-Agha, N., et al. (2016). The aryl hydrocarbon receptor promotesaging phenotypes across species. Sci Rep 6, 19618). Inhibition ofsenescence or activation of proliferation have also been noted byseveral of the miRNAs identified here, including miR-17-5p, miR-93-3p,miR-1260b, miR-106a-5p, miR-767-5p, miR-130a-3p, miR-9-3p, miR-9-5p, andmiR-130b-3p (Borgdorff, V., Lleonart, M. E., Bishop, C. L., Fessart, D.,Bergin, A. H., Overhoff, M. G., and Beach, D. H. (2010). MultiplemicroRNAs rescue from Ras-induced senescence by inhibitingp21(Waf1/Cip1). Oncogene 29, 2262-2271). Together, these data provide acomplementary mechanism by which fetal hGPCs may maintain theircharacteristic progenitor transcriptional state and signature.

Example 7: Adult miRNA Signaling May Repress the ProliferativeProgenitor State and Augur Senescence

The study next inspected the potential miRNA regulatory network withinadult hGPCs (FIG. 6 , Panel E). This implicated five miRNAs controllingfive identified active fetal transcriptional regulators including HDAC2,NFIB, BCLL1A, TEAD2, and HMGA2, whose silencing via miR-4651 haspreviously been shown to inhibit proliferation (Han, X., Yang, R., Yang,H., Cao, Y., Han, N., Zhang, C., Shi, R., Zhang, Z, Fan, Z. (2020).miR-4651 inhibits proliferation of gingival mesenchymal stem cells byinhibiting HMGA2 under nifedipine treatment. Int J Oral Sci 12, 10).This cohort of miRNAs were predicted to operate in parallel to adulttranscriptional repressors in inhibiting expression of genes involved inmaintaining the GPC progenitor state including PDGFRA, PTPRZ1, ZBTB18,SOX6, EGFR, and NRXN1. Furthermore, the adult miRNA environment waspredicted to repress numerous genes known to induce a proliferativestate or to delay senescence, including LMNB1 (Freund, A., Laberge, R.M., Demaria, M., and Campisi, J. (2012). Lamin B1 loss is asenescence-associated biomarker. Mol Biol Cell 23, 2066-2075),PATZ1(Cho, J. H., Kim, M. J., Kim, K. J., and Kim, J. R. (2012). POZ/BTBand AT-hook-containing zinc finger protein 1 (PATZ1) inhibitsendothelial cell senescence through a p53 dependent pathway. Cell DeathDiffer 19, 703-712), GADD45A (Hollander, M. C., Sheikh, M. S., Bulavin,D. V., Lundgren, K., Augeri-Henmueller, L., Shehee, R., Molinaro, T. A.,Kim, K. E., Tolosa, E., Ashwell, J. D., et al. (1999). Genomicinstability in Gadd45a-deficient mice. Nat Genet 23, 176-184), YAP1 andTEAD1 (Xie, Q., Chen, J., Feng, H., Peng, S., Adams, U., Bai, Y., Huang,L., Li, J., Huang, J., Meng, S., et al. (2013). YAP/TEAD- mediatedtranscription controls cellular senescence. Cancer Res 73, 3615-3624.),CDK1 (Diril, M. K., Ratnacaram, C. K., Padmakumar, V. C., Du, T.,Wasser, M., Coppola, V., Tessarollo, L., and Kaldis, P. (2012).Cyclin-dependent kinase 1 (Cdk1) is essential for cell division andsuppression of DNA re-replication but not for liver regeneration. ProcNatl Acad Sci U S A 109, 3826-3831), TPX2 (Rohrberg, J., Van de Mark,D., Amouzgar, M., Lee, J. V., Taileb, M., Corella, A., Kilinc, S.,Williams, J., Jokisch, M. L., Camarda, R., et al. (2020). MYCDysregulates Mitosis, Revealing Cancer Vulnerabilities. Cell Rep 30,3368- 3382), S1PR1 (Liu, Y., Zhi, Y., Song, H., Zong, M., Yi, J., Mao,G., Chen, L., and Huang, G. (2019). S1PR1 promotes proliferation andinhibits apoptosis of esophageal squamous cell carcinoma throughactivating STAT3 pathway. Journal of Experimental & Clinical CancerResearch 38, 369), RRM2 (Aird, K. M., Zhang, G., Li, H., Tu, Z., Bitler,B. G., Garipov, A., Wu, H., Wei, Z., Wagner, S. N., Herlyn, M., et al.(2013). Suppression of nucleotide metabolism underlies the establishmentand maintenance of oncogene-induced senescence. Cell Rep 3, 1252-1265),CCND2 (Bunt, J., de Haas, T. G., Hasselt, N. E., Zwijnenburg, D. A.,Koster, J., Versteeg, R., and Kool, M. (2010). Regulation of cell cyclegenes and induction of senescence by overexpression of OTX2 inmedulloblastoma cell lines. Mol Cancer Res 8, 1344-1357), SGO1(Murakami-Tonami, Y., Ikeda, H., Yamagishi, R., Inayoshi, M., Inagaki,S., Kishida, S., Komata, Y., Jan, K., Takeuchi, I., Kondo, Y., et al.(2016). SGO1 is involved in the DNA damage response in MYCN-amplifiedneuroblastoma cells. Scientific Reports 6, 31615), MCM4 and MCM6 (Mason,D. X., Jackson, T. J., and Lin, A. W. (2004). Molecular signature ofoncogenic ras-induced senescence. Oncogene 23, 9238-9246.), ZNF423(Hernandez-Segura, A., de Jong, T. V., Melov, S., Guryev, V., Campisi,J., and Demaria, M. (2017). Unmasking Transcriptional Heterogeneity inSenescent Cells. Current biology: CB 27, 2652-2660), PHB (Piper, P. W.,Jones, G. W., Bringloe, D., Harris, N., MacLean, M., and Mollapour, M.(2002). The shortened replicative life span of prohibitin mutants ofyeast appears to be due to defective mitochondrial segregation in oldmother cells. Aging cell 1, 149-157), WLS (Poudel, S. B., So, H. S.,Sim, H. J., Cho, J. S., Cho, E. S., Jeon, Y. M., Kook, S. H., and Lee,J. C. (2020). Osteoblastic Wntless deletion differentially regulates thefate and functions of bone marrow-derived stem cells in relation to age.Stem Cells.), and ZMAT3 (Kim, B. C., Lee, H. C., Lee, J. J., Choi, C.M., Kim, D. K., Lee, J. C., Ko, Y. G., and Lee, J. S. (2012). Wiglprevents cellular senescence by regulating p21 mRNA decay throughcontrol of RISC recruitment. EMBO J 31, 4289-4303). More directly,induction of senescence or inhibition of proliferation has been linkedto the upregulation of miR-584-5p (Li, Q., Li, Z., Wei, S., Wang, W.,Chen, Z., Zhang, L., Chen, L., Li, B., Sun, G., Xu, J., et al. (2017).Overexpression of miR-584-5p inhibits proliferation and inducesapoptosis by targeting WW domain-containing E3 ubiquitin protein ligase1 in gastric cancer. J Exp Clin Cancer Res 36, 59), miR-193a-5p (Chen,J., Gao, S., Wang, C., Wang, Z., Zhang, H., Huang, K., Zhou, B., Li, H.,Yu, Z., Wu, J., et al. (2016). Pathologically decreased expression ofmiR-193a contributes to metastasis by targeting WT1-E-cadherin axis innon-small cell lung cancers. J Exp Clin Cancer Res 35, 173), miR-548ac(Song, F., Yang, Y., and Liu, J. (2020). MicroRNA-548ac inducesapoptosis in laryngeal squamous cell carcinoma cells by targetingtransmembrane protein 158. Oncol Lett 20, 69), miR-23b-3p(Campos-Viguri, G. E., Peralta-Zaragoza, O., Jimenez-Wences, H.,Longinos-Gonzalez, A. E., Castanon-Sanchez, C. A., Ramirez-Carrillo, M.,Camarillo, C. L., Castaneda-Saucedo, E., Jimenez-Lopez, M. A.,Martinez-Carrillo, D. N., et al. (2020). MiR-23b-3p reduces theproliferation, migration and invasion of cervical cancer cell lines viathe reduction of c-Met expression. Sci Rep 10, 3256), miR-140-3p (Wang,M., Wang, X., and Liu, W. (2020a). MicroRNA-130a-3p promotes theproliferation and inhibits the apoptosis of cervical cancer cells vianegative regulation of RUNX3. Mol Med Rep 22, 2990- 3000), andmiR-330-3p (Wang, Y., Chen, J., Wang, X., and Wang, K. (2020b).miR-140-3p inhibits bladder cancer cell proliferation and invasion bytargeting FOXQ1. Aging 12, 20366- 20379). Taken together, these dataimplicate these miRs as active participants in maintenance of theprogenitor state in fetal hGPCs, and their modulation as a likelymechanism by which adult hGPCs assume their signatory gene expressionprofile.

Example 8: Transcription Factor Regulation of miRNAs Establishes andConsolidates GPC Identity

The study next sought to predict the upstream regulation ofdifferentially expressed miRNAs in fetal and adult GPCs by querying theTransmiR transcription factor miRNA regulation database (Tong, et al.(2019). TransmiR v2.0: an updated transcription factor-microRNAregulation database. Nucleic Acids Res 47, D253-D258). This approachpredicted regulation of 54 of 56 of age-specific GPC miRNAs via 66transcription factors that were similarly determined to be significantlydifferentially expressed between fetal and adult GPCs. Interestingly,the top four predicted miRNA-regulating TFs were all MYC-associatedfactors including MAX, MYC itself, E2F6, and the fetal enriched MYCassociated zinc finger protein, MAZ, targeting 36, 33, 30, and 28 uniquedifferentially expressed miRNAs respectively.

Inspection of proposed relationships in the context of the 12 TFcandidates indicated a large number of fetal hGPC-enriched miRNAs thatwere predicted to be targeted by both fetal activators and adultrepressors, whereas those miRNAs enriched in adult GPCs were moreuniquely targeted. MYC was predicted to drive the expression of numerousmiRNAs in fetal GPCs, many of which were predicted to be repressed inadulthood via E2F6, MAX or both. miR-130a-3p in particular was predictedto be targeted by MYC, MAX, and E2F6, in addition to activation viaTEAD2. Notably among validated TF-miRNA interactions in other celltypes, the upregulation of the rejuvenating miR-17-5p by MYC, and itsrepression by MAX (Du, et al. (2014b). miR-17 extends mouse lifespan byinhibiting senescence signaling mediated by MKP7. Cell Death Dis 5,e1355), has been reported. Similarly, the parallel activation of theproliferative miR-130-3p by MYC or TEAD2 and YAP1 (Shen, et al. (2015).A miR-130a-YAP positive feedback loop promotes organ size andtumorigenesis. Cell Res 25, 997-1012), has been reported, as has theactivation of both arms of miR-9 by MYC (Ma, L., et al. (2010a). miR-9,a MYC/MYCN-activated microRNA, regulates E-cadherin and cancermetastasis. Nat Cell Biol 12, 247-256), which decreases witholigodendrocytic maturity (Lau, P., et al. (2008). Identification ofdynamically regulated microRNA and mRNA networks in developingoligodendrocytes. J Neurosci 28, 11720-11730).

In adult GPCs, enriched miRNAs predicted to be regulated by oursignificantly enriched TF cohort were more likely to be only targeted byan adult activator of fetal repressor with only miR-151a-5p and miR-468′7-3p, a predicted inhibitor of HMGA2, being targeted in oppositionby STAT3 versus BCL11A and EZH2 respectively. Beyond this, miR-1268b waspredicted to be inhibited by both EZH2 and HDAC2 in parallel. Notably,key oligodendrocytic microRNA, miR-219a-2-3p was predicted to remaininhibited in fetal GPCs via EZH2, whereas STAT3 likely drives theexpression of 7 other miRs independently. Interestingly, STAT3, whoseincreased activity has been linked to senescence (Kojima, et al. (2013).IL-6-STAT3 signaling and premature senescence. JAKSTAT 2, e25763), wasalso predicted to drive the expression of a cohort of miRNAsindependently associated with the induction of senescence, includingmiR- 584-5p, miR-330-3p, miR-23b-3p, and miR-140-3p.

Through integration of transcriptional and miRNA profiling, pathwayenrichment analyses, and target predictions, we propose a model of humanGPC aging whereby fetal hGPCs maintain progenitor gene expression,activate proliferative programs, and prevent senescence, whilerepressing oligodendrocytic and senescent gene programs bothtranscriptionally, and post-transcriptionally via microRNA. With adultmaturation and the passage of time as well as of population doublings,hGPCs begin to upregulate repressors of these fetal progenitor-linkednetworks, while also activating programs to further a progressively moredifferentiated and ultimately senescent phenotype. Example 9: Expressionof BCL11A in the Brain of Chimeric Animal Model Results in Proliferationof BCL11A Expressing GPCs in the Host

By analyzing RNA sequencing data from fetal and adult human glialprogenitor cells (GPCs) sorted from fresh tissue samples, the studyidentified several transcription factors as central in gene regulatorynetworks that distinguish f “young” vs “old” GPCs. Among these, thetranscriptional repressor BCL11A (B cell CLL/lymphoma 11A) was one ofthe most prominently differentially expressed genes, high in fetal hGPCsand low in adult cells, suggesting its role in preserving the fetal hGPCphenotype. BCL11A had never been known to play such a role in thecentral nervous system, or in the regulation of glial progenitor cellexpansion, fate or aging.

To investigate then whether BCL11A could re-initiate or accelerateself-renewal in aged GPCs, the study generated a lentivirus to expressBCL11A and GFP driven by the CBh promoter (hereby referred to asCBh-BCL11A), as well as a green fluorescent protein (GFP)-only controlvirus (CBh-GFP) (FIG. 14 a ). GPCs were generated from the human C27iPSC line, tagged with a red fluorescent-expressing transgene (RFP)(Fig14 b ), then engrafted into the corpus callosum (CC) of Rag1immunodeficient mice on postnatal day 1, as previously described (P1,300k cells/animal) (Windrem at al. J. Neurosci 34, 16153-16161 (2014),Windrem et al., Cell Stem Cell 21, 195-208.e6 (2017)). Chimerized micewere allowed to age for 2 years following engraftment, at which pointthey received stereotactic injection of CBh-BCL11A in the lefthemisphere, and CBh-GFP control virus in the right hemisphere. Virus wasdeposited in the striatum, corpus callosum, and cortex. At either 3 or 6weeks post-injection, mice were either dissected for single-cellanalysis, or perfused with 4% PFA for sectioning andimmunohistochemistry (FIG. 14 c ).

The study confirmed overexpression of BCL11A via both RNA expression invitro (FIG. 14 d ), and protein staining in vivo (FIG. 14 e ). Threeweeks post-infection, the study observed an expansion of the GPCpopulation in the CBh-BCL11A-treated hemisphere, as compared to thecontrol side of the brain (FIG. 15 a ). The study found a robustincrease in the presence of RFP-tagged human cells and OLIG2+ cells ofthe GPC lineage (FIG. 15 b , quantified in 15c), as well as an increasein resident mouse GPCs, identified by the mouse NG2 antigen (FIG. 15 d ,quantified in 15e). This effect was still present at 6 weekspost-infection (FIG. 16 a ), with more RFP+ and OLIG2+ cells detected onthe CBh-BCL11A-infected hemisphere than in the CBh-GFP-treated controls(FIG. 16 b ). At that 6 week timepoint, donor cell dispersal was bothbroad and relatively uniform; no tumors or heterotopias were found.These observations indicated that BCL11A transduction activated bothaged resident human and mouse GPCs alike, to re-initiate mitoticexpansion and migratory colonization of their host brains. Furthermore,the (red) membrane-tagging of the BCL11A-mobilized human donor cellsallowed the morphologies of a large fraction of those in the whitematter to be defined as myelinating oligodendrocytes, suggesting atleast partial reversal of the typical age-associated loss of myelin inthese aged mice.

Herein incorporated by reference is the sequence listing filed with theUSPTO as 1134-086US_Sequence.xml which was created on Oct. 17, 2022, andthe size is 21.8 KB.

While various embodiments have been described above, it should beunderstood that such disclosures have been presented by way of exampleonly and are not limiting. Thus, the breadth and scope of the subjectcompositions and methods should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the components and steps in any sequence which iseffective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A method of inducing rejuvenation in a populationof adult glial progenitor cells, said method comprising: administering,to the population of adult glial progenitor cells, an effective amountof an agent that suppresses one or more transcription factors selectedfrom the group consisting of (i) zinc finger protein 274 (ZNF274), (ii)Myc-associated factor X (MAX), (iii) E2F transcription factor 6 (E2F6),(iv) zinc finger protein Aiolos (IKZF3), and (v) signal transducer andactivator of transcription 3 (STAT3).
 2. A method of treating a subjecthaving a glial cell-related disorder, said method comprising:administering, to the subject, an effective amount of an agent thatsuppresses one or more transcription factors selected from the groupconsisting of (i) zinc finger protein 274 (ZNF274), (ii) Myc-associatedfactor X (MAX), (iii) E2F transcription factor 6 (E2F6), (iv) zincfinger protein Aiolos (IKZF3), and (v) signal transducer and activatorof transcription 3 (STAT3).
 3. The method of claim 2, wherein the glialcell-related disorder is myelin deficiency.
 4. The method of claim 3,wherein the myelin deficiency is associated with a condition selectedfrom the group consisting of multiple sclerosis, neuromyelitis optica,transverse myelitis, optic neuritis, subcortical stroke, diabeticleukoencephalopathy, hypertensive leukoencephalopathy, age-related whitematter disease, spinal cord injury, radiation- or chemotherapy induceddemyelination, post-infectious and post-vaccinial leukoencephalitis,periventricular leukomalacia, pediatric leukodystrophies, lysosomalstorage diseases, congenital dysmyelination, inflammatory demyelination,vascular demyelination, and cerebral palsy.
 5. The method of claim 2,wherein the glial cell-related disorder is a neurodegenerative diseaseselected from the group consisting of Huntington's disease,frontotemporal dementia, Parkinson's disease, multisystem atrophy, andamyotrophic lateral sclerosis.
 6. The method of claim 5, wherein theglial cell-related disorder is Huntington's disease.
 7. The method ofclaim 2, wherein the subject is human and wherein the glial cell-relateddisorder is a neuropsychiatric disorder selected from the groupconsisting of schizophrenia, autism spectrum disorder, and bipolardisorder.
 8. The method of claim 1, wherein the agent comprises anantisense oligonucleotide.
 9. The method of claim 1, wherein the agentcomprises a nucleic acid molecule comprising a nucleic acid sequenceencoding a miRNA, a shRNA or a siRNA.
 10. The method of claim 1, whereinthe agent comprises one or more nucleic acid molecules that comprise afirst nucleic acid sequence encoding a Cas protein and a second nucleicacid sequence encoding a guide RNA.
 11. The method of claim 10, whereinthe first nucleic acid sequence and the second nucleic acid sequence arelocated on the same nucleic acid molecule.
 12. The method of claim 10,wherein the Cas protein is a nuclease dead Cas protein.
 13. The methodof claim 9, wherein the agent is a non-viral expression vector.
 14. Themethod of claim 9, wherein the agent is a viral expression vector. 15.The method of claim 14, wherein the viral expression vector is alentiviral vector.
 16. The method of claim 14, wherein the viralexpression vector is an AAV vector.
 17. The method of claim 9, whereinthe nucleic acid sequence encoding a miRNA, a shRNA or a siRNA, thefirst nucleic acid sequence encoding a Cas protein and the secondnucleic acid sequence encoding a guide RNA are operably linked to aregulatory element.
 18. The method of claim 17, wherein the regulatoryelement is a glial cell-specific promoter.
 19. The method of claim 17,wherein the regulatory element is an inducible promoter.
 20. The methodof claim 19, wherein the inducible promoter is a tet-on or tet-offpromoter.